Sodium channel protein type iii alpha-subunit splice variant

ABSTRACT

The present invention is directed to a splice variant of a human sodium channel alpha subunit and methods and compositions for making and using the same.

This application claims the benefit of priority of prior-filed U.S.provisional application No. 60/654,019, which was filed on Feb. 17, 2005and is incorporated herein by reference in its entirety.

BACKGROUND

1. Field of the Invention

The present invention is generally directed to voltage-gated sodiumchannel Na_(V) 1.3 splice variants. The invention further describesmethods and compositions for the stable expression of such splicevariants and methods of use of such compositions for identifyingcompounds that modulate the activity of sodium channels.

2. Background of the Related Art

The electrical activity of neuronal and muscle cells are governed by theactivity of sodium channels on the plasma membrane of such cells. Rapidentry of sodium ions into the cell through such a channel causesdepolarization of the membrane and generation of an action potential.Entry of sodium ions through sodium channels in response to a voltagechange on the plasma membrane in excitable cells plays a functional rolein the excitation of neurons in the central nervous system and theperipheral nervous system.

Sodium channels are voltage-gated transmembrane proteins that form ionchannels within the membrane and have been the target of significantpharmocologic study, due to their potential role in a variety ofpathological conditions. These sodium channels are responsible for thecellular uptake of sodium during the transmission of an electricalsignal in cell membranes. The channels are members of a multigene familyof proteins and are typically composed of a number of subunits.Typically, the pore of the channel is formed by the α-subunit and thereare four accessory β-subunits, termed β1, β2, β3 and β3.

The β-subunits are involved in the modulation of the activity of sodiumchannel but the α-subunit is all that is required for the channel toform a functional ion pore. Co-expression of the β-subunits with theα-subunit has been shown to produce a more positive membrane potential.Further not all of the β-subunits are required, for example, it has beenshown that the β3-subunits alone is sufficient to cause an increase insodium current (Qu et al., Mol. Cell. Neurosci., 18(5):570-80, 2001).

The amino acid sequence of the sodium channel has been evolutionarilyconserved. The channel is comprised of a signal polypeptide containingfour internal repeats (domains I-IV). Each domain folds into sixtransmembrane α-helices or segments, of which five are hydrophobic andone is a highly-charged domain containing lysine and arginine residues(S4 segment). The highly-charged S4 segment is involved in the voltagegating properties of the sodium channel. The positively-charged sidechains of the amino acids of the S4 segment are thought to be pairedwith the negatively-charged side chains of the other five segments suchthat upon membrane depolarization there is a shift in the position ofone of the helices relative to the other resulting in an opening of thechannel.

There are numerous variants of sodium channel α-subunit. These variantsmay be classified according to their sensitivity to tetrodotoxin (TTX).Those subunits that are inhibited by nanomolar quantities of TTX areclassified as tetrodotoxin-sensitive channels, whereas those thatrequire at least micromolar quantities of TTX for inhibition arereferred to as tetrodotoxin-insensitive (1-5 micromolar). Those channelsthat require greater that 100 micromolar quantities of the TTX aretermed tetrodotoxin-resistant. TTX is a toxin that blocks the conductionof nerve impulses along the axons and leads to paralysis. It binds tosodium channels and blocks the flow of sodium ions. It is believed thatthe positively-charged group of the toxin interacts with anegatively-charged carboxylate at the mouth of the channel on theextracellular side of the membrane thereby blocking the conductance ofthe pathway.

It has been noted that following nerve injury there is hyperexcitability(or an increased rate of spontaneous impulse firing in neurons) inperipheral sensory ganglia. It has been suggested that thishyperexcitability in neurons is due to altered sodium channel expressionin some chronic pain syndromes (Tanaka et al., Neuroreport 1998; 9 (6):967-72). Increased numbers of sodium channels leading to inappropriate,repetitive firing of the neurons have been reported in the tips ofinjured axons in various peripheral nervous tissues such as the DRG,which relay signals from the peripheral receptors to the central nervoussystem. Indeed, it has been noted that there is an increase inexpression of an α1 Na_(V) 1.3 subunit in axotomized DRG neuronstogether with elevated levels of α1 Na_(V)1.1 and α1 Na_(V)1.2 mRNAs(Waxman et al, Brain Res Mol Brain Res 1994; 22 (1-4): 275-89).

The peripheral input that drives pain perception is thought to dependupon the presence of functional voltage-gated sodium channels inperipheral nerves. It has been noted that there is a positivecorrelation between increased sodium channel expression in peripheralnerves. Some studies have also shown increased expression in associationwith neuropathic pain. In particular, it has been recognized that acute,inflammatory, and neuropathic pain can all be attenuated or abolished bylocal treatment with sodium channel blockers such as lidocaine.Remarkably, two voltage-gated sodium channel genes (Nav1.8 and Nav1.9)are expressed selectively in damage-sensing peripheral neurons, while athird channel (Nav1.7) is found predominantly in sensory and sympatheticneurons. An embryonic channel (Nav1.3) is also upregulated in damagedperipheral nerves and associated with increased electrical excitabilityin neuropathic pain states. Using antisense and knock-out studies, ithas been shown that these sodium channels play a specialized role inpain pathways, and pharmacological studies (Wood et al., J Neurobiol.,61(1):55-71, 2004).

Most patients with traumatic spinal cord injury (SCI) report moderate tosevere chronic pain that is refractory, or only partially responsive, tostandard clinical interventions (Balazy, Clin J Pain 8: 102-110, 1992;Turner et al., Arch Phys Med Rehabil 82: 501-509, 2001). Experimentalcontusion SCI in rodents can produce long-lasting central neuropathicpain (Hulsebosch et al., J Neurotrauma 17: 1205-1217, 2000; Lindsey etal., Neurorehabil Neural Repair 14: 287-300, 2000; Hains et al.,Neuroscience 116: 1097-1110, 2001; Mills et al., J Neurotrauma 18:743-756, 2001). In spinally injured animals, alterations inelectrophysiologic properties of dorsal horn neurons (Hao et al., Pain45: 175-185, 1991; Yezierski and Park, Neurosci Lett 157: 115-119, 1993;Drew et al., Brain Res 893: 59-69, 2001; Hains et al., Neuroscience 116:1097-1110, 2003a; Hains et al., Brain Res 970: 238-241, 2003b) arethought to contribute to changes in somatosensory responsiveness.

The TTX-sensitive Nav1.3-sodium channel is expressed at relatively highlevels in embryonic dorsal root ganglion (DRG) neurons but is barelydetectable in adult DRG neurons and its expression is decreased in theadult spinal cord and CNS throughout development. However, theexpression of Nav1.3 mRNA and protein is markedly upregulated in DRGneurons of adult rats after axotomy of peripheral projections, afterchronic constriction injury, and after tight spinal nerve ligation. Thisproduces a rapidly repriming TTX-S current that permits neuronal firingat higher than normal frequencies. It has been shown that an increase inexpression of Nav1.3, similar to the changes in DRG neurons afterperipheral axotomy, takes place in lumbar dorsal horn sensory neuronsafter SCI. It has further been shown that knock-down or reduction ofexpression of Nav1.3 mRNA and protein results in a reduction inhyperexcitability of dorsal horn sensory neurons and pain-relatedbehaviors in animals (Hains et al., J. Neurosci., 23(26):0881-8892,2003).

There are various sodium channels that remain to be characterized.Identification of such channels will facilitate further studies andidentification and characterization of further isotype-specificantagonists of sodium channel blockers. Such sodium channel blockers orantagonists will be useful in the management of pain. Preferably, suchanalgesic agents are such that treatment of pain is facilitated withouthaving deleterious side effects due to cardiac, central nervous systemor neuromuscular complications.

SUMMARY OF THE INVENTION

The present invention is directed to a splice variant of a human sodiumchannel alpha subunit and methods and compositions for making and usingthe same. More specifically, one embodiment of the invention is directedto an isolated recombinant nucleic acid encoding a human sodium channelNaV1.3 polypeptide wherein the polypeptide is encoded by the nucleicacid sequence presented in SEQ ID NO: 1.

Another embodiment of the invention describes an isolated recombinantnucleic acid encoding a human sodium channel NaV1.3 recombinant proteinhaving the amino acid sequence of SEQ ID NO: 2. Also contemplated hereinis an isolated recombinant nucleic acid comprising the sequencepresented in SEQ ID NO: 1, the mature protein coding portion thereof, ora complement thereof. One preferred embodiment of the inventioncontemplates an isolated recombinant nucleic acid encoding a polypeptideof SEQ ID NO: 2. The nucleic acids described herein may be genomic DNA,cDNA, or RNA.

Conservative variants of the sequences of the present invention areparticularly contemplated, for example, the invention is directed to anisolated nucleic acid comprising a nucleotide sequence encoding apolypeptide that is a conservative variant of the amino acid sequenceset forth in SEQ ID NO:2, wherein the variant encodes a sodium channelα-subunit with the proviso that residue 208 of SEQ ID NO:2 is anaspartic acid residue and an insert of 33 amino acids is found afterresidue 623, as defined in SEQ ID NO:2.

Expression constructs that comprise an isolated nucleic acid encoding aprotein having an amino acid sequence of SEQ ID NO:2 or the matureprotein portion thereof wherein the mature protein region comprises anaspartic acid residue at the residue that corresponds to amino acidresidue 208 in SEQ ID NO:2, an insert of 33 amino acids is found afterresidue 623, as defined in SEQ ID NO:2 and a promoter operably linked tothe polynucleotide also form part of the invention. In specificembodiments, the expression construct is such that the nucleic acidcomprises a mature protein coding sequence as set forth in SEQ ID NO:1.The expression construct is an expression construct selected from thegroup consisting of an adenoassociated viral construct, an adenoviralconstruct, a herpes viral expression construct, a vaccinia viralexpression construct, a retroviral expression construct, a lentiviralexpression construct and a naked DNA expression construct.

Also part of the invention are recombinant host cell stably transformedor transfected with a nucleic acid or an expression construct of theinvention in a manner that allows the expression in the host cell of aprotein of SEQ ID NO:2. Preferably, the nucleic acid transforming thehost cell comprises a mature protein encoding sequence as set forth inSEQ ID NO:1, wherein the mature protein encoded by the sequence is asodium channel NaV1.3 polypeptide that has an aspartic acid residue atthe amino acid that corresponds to amino acid residue 208 of SEQ ID NO:2and an insert of 33 amino acids is found after residue 623, as definedin SEQ ID NO:2. Recombinant host cells stably transformed or transfectedwith an expression construct of the invention in a manner allowing theexpression in the host cell of a protein product of the expressionconstruct also are contemplated.

The host cells may be mammalian, a bacterial, yeast cells, or insectcells. It may be advantageous that the recombinant host cells producedby the invention further express one or more β-subunits of a sodiumchannel selected from the group consisting of β1, β2, β3 and β4. Inspecific embodiments, the host cell is a HEK293 cell line.

The invention further provides an isolated and purified proteincomprising an amino acid sequence selected from the group consisting ofan amino acid sequence set forth in SEQ ID NO:2 and the mature proteinportion of SEQ ID NO:2, wherein the mature protein portion comprises anaspartic acid residue at the amino acid residue that corresponds toamino acid residue 208 of SEQ ID NO:2 and an insert of 33 amino acids isfound after residue 623, as defined in SEQ ID NO:2. In particularembodiments, the isolated and purified protein comprises an amino acidsequence that is 99% identical to the complete sequence set forth in SEQID NO:2. In other embodiments, the isolated and purified proteincomprises an amino acid sequence that is 95% identical to the completesequence of SEQ ID NO:2 and contains a 33 amino acid insert of SEQ IDNO:3.

The invention also comprises a diagnostic kit for detecting a nucleicacid that encodes a sodium channel α-subunit polypeptide, thepolypeptide being encoded by the sequence presented in SEQ ID NO: 1,comprising an isolated nucleic acid probe complementary to the completesequence of SEQ ID NO: 1, and means for containing the nucleic acid.

Methods of identifying a modulator of a human sodium channel α-subunitexpression or activity are contemplated wherein the modulator isidentified by a method comprising the steps of contacting a cell thatexpresses a nucleic acid of SEQ ID NO:1 with the candidate modulatorsubstance; monitoring the expression or ion channel activity of aprotein of SEQ ID NO:2; and comparing the expression or ion channelactivity of a protein of SEQ ID NO:2 in the presence and absence of thecandidate substance; wherein an alteration in the expression or ionchannel activity of a protein of SEQ ID NO:2 indicates that thesubstance is a modulator of human sodium channel α-subunit expression oractivity. The modulator of human sodium channel α-subunit expression oractivity may be a small molecule ion channel blocker or inhibitor, anoligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, anRNA oligonucleotide, an RNA oligonucleotide having at least a portion ofthe RNA oligonucleotide capable of hybridizing with RNA to form anoligonueleotide-RNA duplex, or a chimeric oligonucleotide.

The invention also provides methods of identifying a test compound thatbinds to a sodium channel comprising providing a cell that expresses asodium channel having a sequence of SEQ ID NO:2; contacting the hostcell with the test compound and determining the binding of the testcompound to the sodium channel; and comparing the binding of the testcompound to the host cell determined in step (b) to the binding of thetest compound with a cell that does not express a sodium channel.

Also provided is an assay for identifying a test compound that modulatesthe activity of a sodium channel comprising providing a host cell thatexpresses a functional sodium channel comprising at least onepolypeptide comprising the amino acid sequence of SEQ ID NO: 2,contacting the host cell with a test compound under conditions thatwould activate sodium channel activity of the functional sodium channelin the absence of the test compound; and determining whether the hostcell contacted with the test compound exhibits a modulation in activityof the functional sodium channel. In particular embodiments, the hostcell has been genetically engineered to express or overexpress thefunctional sodium channel. In other embodiments, the host cell has beengenetically engineered by the introduction into the cell of a nucleicacid molecule having a nucleotide sequence encoding a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 2.Preferably, the host cell has been genetically engineered to upregulatethe expression of a nucleic acid encoding a polypeptide comprising theamino acid sequence of SEQ ID NO: 2. In particular embodiments, theupregulated nucleic acid is endogenous to the host cell. Preferably, themodulation of the functional sodium channel activity is an inhibition ofthat activity.

A method of producing a purified human sodium channel α-subunit proteinalso is provided, the method comprising preparing an expressionconstruct comprising a nucleic acid of SEQ ID NO:1 operably linked to apromoter; transforming or transfecting a host cell with the expressionconstruct in a manner effective to allow the expression of a proteinhaving an amino acid sequence of SEQ ID NO:2, or the mature proteinportion thereof in the host cell; culturing the transformed ortransfected cell under conditions to allow the production of the proteinby the transformed or transfected host cell; and isolating the humansodium channel α-subunit protein from the host cell.

Other embodiments contemplate treatment of a disorder by administeringto a subject in need thereof a pharmaceutical composition that comprisesa compound identified according to the methods described herein and apharmaceutically acceptable carrier, excipient or diluent.

Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, because various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further illustrate aspects of the present invention. Theinvention may be better understood by reference to the drawings incombination with the detailed description of the specific embodimentspresented herein.

FIGS. 1A through 1F shows the sequence alignment of the sequence of thepresent invention (SEQ ID NO:2) with sequences (SEQ ID NO:4; SEQ IDNO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8) identified from Genbank.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Human Na_(V)1.3 is a voltage-gated sodium channel α-subunit whoseexpression has been shown to be upregulated in neurons that have beensubjected to various injuries such as e.g., after axotomy of peripheralprojections, after chronic constriction injury, and after tight spinalnerve ligation. Reduction or knockout Nav1.3 alleviates the painassociated with such injuries. Thus, therapies designed to knock-out orblock the action of Nav1.3 are important in alleviating neuropathy. Thepresent application is directed to a novel splice variant of the α1subunit of human Nav 1.3.

The splice variant of the human Na_(V)1.3 channel α subunit of thepresent invention was identified from human spinal cord RNA using RT-PCRto amplify the message in three overlapping fragments as described inthe Examples herein below. The three fragments were then ligatedtogether to create the full-length sequence. The cDNA clone isolated isa novel splice variant that differs from previously reported Nav1.3cDNAs. This Nav1.3 cDNA was used to express human Nav1.3 in culturedcell lines. The Nav 1.3-expressing line was used in high-throughputscreening to identify antagonists of Nav 1.3 and has been used tocharacterize the activity of such agents against the channel. Methodsand compositions for making and using the splice variant of the presentinvention are described in further detail below.

Polypeptide and Fragments Thereof.

According to the present invention, there has been identified a genethat encodes a novel splice variant of human Nav1.3. It is contemplatedthat this gene and the protein encoded by the same may be used instudies of sodium channels and in the identification of modulatorsthereof. In this regard, it is noted that sodium channel α-subunits arewell known to those of skill in the art and have been described e.g., inU.S. Pat. No. 6,479,259; U.S. Pat. No. 6,335,172; U.S. Pat. No.6,184,349; U.S. Pat. No. 6,060,271; U.S. Pat. No. 6,030,810; U.S. Pat.No. 5,892,018; U.S. Pat. No. 5,776,859; U.S. Pat. No. 5,693,756; U.S.Pat. No. 5,437,982; U.S. Pat. No. 5,380,836. Each of the foregoingpatents are incorporated herein by reference as providing specificteaching of how to make and use sodium channel proteins and nucleicacids that encode the same. The methods taught therein for using suchcompositions to identify therapeutic agents, e.g., sodium channelblockers or even β-subunits that are modulators of sodium channelα-subunits may readily be adapted using the protein and nucleic acidcompositions of the present invention. As discussed above, Nav1.3 sodiumchannels are involved in mediating pain associated with neuronal injury.As such, it is contemplated that it will be will be desirable toinhibit, decrease, ablate, reduce or otherwise diminish the expressionof the Nav1.3 gene or the activity of the protein product of the geneexpression described herein. It is contemplated that inhibition ofactivity of the encoded protein or the expression of this gene will havea beneficial effect in treating pain. Inhibition of the gene expressionmay even be helpful in regenerative studies or overcoming thedeleterious effects of spinal cord injury.

In the therapeutic aspects, guidance may be gained from the functionaland therapeutic aspects of sodium channels described in e.g., in U.S.Pat. No. 6,479,259; U.S. Pat. No. 6,335,172; U.S. Pat. No. 6,184,349;U.S. Pat. No. 6,060,271; U.S. Pat. No. 6,030,810; U.S. Pat. No.5,892,018; U.S. Pat. No. 5,776,859; U.S. Pat. No. 5,693,756; U.S. Pat.No. 5,437,982; U.S. Pat. No. 5,380,836 been recognized as being involvedin pain and have been used as targets for therapy. Sodium channelblockers or modulators have been described e.g., in U.S. Pat. No.6,756,400; U.S. Pat. No. 6,646,012; U.S. Pat. No. 6,613,345; U.S. Pat.No. 6,607,741; U.S. Pat. No. 6,559,154; U.S. Pat. No. 6,479,498 (eachincorporated by reference) and such patents provide guidance as tomethods and compositions for identification of additional suchtherapeutic agents once new targets such as the Nav1.3 splice variant ofthe present invention, are identified. While treatment of pain and thelike will involve inhibition or blocking of Nav1.3 activity, it iscontemplated that in certain embodiments, it may be desirable toincrease the expression of Nav1.3. For example, in specific embodiments,it would be desirable to increase, augment or otherwise supplementendogenous Nav1.3 expression and/or activity in commercial orexperimental endeavors where it would be desirable to produce animalmodels or cells that have an increased Nav1.3 expression and arephenotypically models for neuropathic pain.

The Nav1.3 splice variant encoding gene has been cloned by the presentinventors and is taught herein to have a nucleic acid sequence as shownin SEQ ID NO:1. The coding region of this gene encodes a protein of SEQID NO:2. It is noted that the encoded protein has an aspartic acidresidue at an amino acid that corresponds to residue number 208 of SEQID NO:2. A sequence alignment of the sequence of SEQ ID NO:2 with othersodium channel alpha subunit proteins is shown in FIG. 1. As can be seenin FIG. 1, there is an aspartic acid residue at amino acid residue 208.Further, SEQ ID NO:2 comprises an insert of NVSQASMSSRMVPGLPANGKMHSTVDCNGVVSL that is not present in other sodium channelalpha subunits examined in FIG. 1. This 33 amino acid insert that startsat residue 624 of the NAV1.3 splice variant of the present invention.This region is the linker between domain 1 and domain 2 of the sodiumchannel alpha subunit. Protein kinase A phosphorylation sites of thesodium channel are located in this region and are close to this splicesite. The sodium channel activity of the protein of SEQ ID NO:2 or avariant thereof that contains aspartic acid at an amino acid residuethat corresponds to amino acid 208 of the Nav1.3 splice variant of thepresent invention may be readily tested using techniques well known tothose of skill in the art.

In addition to the entire Nav1.3 protein molecule of SEQ ID NO:2, decompositions of the present invention also may employ fragments of thepolypeptide of SEQ ID NO:2 that retain the ability/activity to form asodium channel and retain an aspartic acid at a residue that correspondsto amino acid residue 208 of SEQ ID NO:2. Fragments, including theN-terminus or C terminus of the molecule may be generated by geneticengineering of translation start or stop sites within the coding region(discussed below). Alternatively, treatment of the Nav1.3 splice variantprotein molecule with proteolytic enzymes, (proteases), can produce avariety of N-terminal, C-terminal and internal fragments. Examples offragments may include contiguous residues of the Nav1.3 splice variantprotein sequence of SEQ ID NO:2 of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65,75, 80, 85, 90, 95, 100, or more amino acids in length. Such fragmentspreferably retain one or more of the biological activities of Nav1.3protein and/or retain an immunological (antigenic) property of Nav1.3protein. These fragments may be purified according to known methods,such as precipitation (e.g., ammonium sulfate), HPLC, ion exchangechromatography, affinity chromatography (including immunoaffinitychromatography) or various size separations (sedimentation, gelelectrophoresis, gel filtration).

When the present application refers to the function of Nav1.3 splicevariant protein or “wild-type” activity, it is meant that the moleculein question has the ability to form a sodium channel in a plasmamembrane fraction. The Nav1.3 protein of the present invention has asequence of SEQ ID NO:2. An assessment of the particular molecules thatpossess such activities may be achieved using standard assays familiarto those of skill in the art. For example, the immunological studieswill readily reveal whether the Nav1.3 splice variant binds toantibodies directed against Nav1.3 or other sodium channel alphasubunits. Such antibodies are known to those of skill in the art and maybe readily generated using routine methods.

In certain embodiments, Nav1.3 protein analogs and variants may beprepared and will be useful in a variety of applications. Amino acidsequence variants of the polypeptide can be substitutional, insertionalor deletion variants. Deletion variants lack one or more residues of thenative protein which are not essential for function or immunogenicactivity. A common type of deletion variant is one lacking secretorysignal sequences or signal sequences directing a protein to bind to aparticular part of a cell. Insertional mutants typically involve theaddition of material at a non-terminal point in the polypeptide. Thismay include the insertion of an immunoreactive epitope or simply asingle residue. Terminal additions, also called fusion proteins, arediscussed below.

Substitutional variants typically exchange one amino acid of the wildtype for another at one or more sites within the protein, and may bedesigned to modulate one or more properties of the polypeptide, such asstability against proteolytic cleavage, without the loss of otherfunctions or properties. Substitutions of this kind preferably areconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

In order to construct such mutants, one of skill in the art may employwell known standard technologies. Specifically contemplated areN-terminal deletions, C-terminal deletions, internal deletions, as wellas random and point mutagenesis.

N-terminal and C-terminal deletions are forms of deletion mutagenesisthat take advantage for example, of the presence of a suitable singlerestriction site near the end of the C- or N-terminal region. The DNA iscleaved at the site and the cut ends are degraded by nucleases such asBAL31, exonuclease III, DNase I, and S1 nuclease. Rejoining the two endsproduces a series of DNAs with deletions of varying size around therestriction site. Alternatively, deletions can be generated usingpolymerase chain reaction (PCR) amplification of cDNAs using primersthat exclude regions of the coding sequence corresponding to the desiredpolypeptide deletion. Proteins expressed from such mutants can beassayed for appropriate activity as voltage-gated sodium channels, asdescribed throughout the specification. Similar techniques may beemployed for internal deletion mutants by using two suitably placedrestriction sites, thereby allowing a precisely defined deletion to bemade, and the ends to be religated as above. as above. Similarly, PCRcan be used to amplify the sequences flanking the internal deletion andthen ligation used as described above to generate the DNA clonecontaining the desired deletion.

Also contemplated are partial digestion mutants. In such instances, oneof skill in the art would employ a “frequent cutter”, which cuts the DNAin numerous places depending on the length of reaction time. Thus, byvarying the reaction conditions it will be possible to generate a seriesof mutants of varying size, which may then be screened for activity.

A random insertional mutation may also be performed by cutting the DNAsequence with a DNase I, for example, and inserting a stretch ofnucleotides that encode, 3, 6, 9, 12 etc., amino acids and religatingthe end. Once such a mutation is made the mutants can be screened forvarious activities presented by the wild-type protein.

Point mutagenesis also may be employed to identify with particularitywhich amino acid residues are important in particular activitiesassociated with Nav1.3 splice variant protein. Thus, one of skill in theart will be able to generate single base changes in the DNA strand toresult in an altered codon and a missense mutation.

The amino acids of a particular protein can be altered to create anequivalent, or even an improved, second-generation molecule. Suchalterations contemplate substitution of a given amino acid of theprotein without appreciable loss of interactive binding capacity withstructures such as, for example, antigen-binding regions of antibodiesor binding sites on substrate molecules or receptors. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid substitutions can bemade in a protein sequence, and its underlying DNA coding sequence, andnevertheless obtain a protein with like properties. Thus, variouschanges can be made in the DNA sequences of genes without appreciableloss of their biological utility or activity, as discussed below. Codontables that show the codons that encode particular amino acids are wellknown to those of skill in the art. In making changes to the sequence ofSEQ ID NO:2, the hydropathic index of amino acids may be considered,which contributes to the secondary structure of the resultant protein,which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like (Kyte & Doolittle, J. Mol. Biol., 157(1):105-132,1982, incorporated herein by reference). Generally, amino acids may besubstituted by other amino acids that have a similar hydropathic indexor score and still result in a protein with similar biological activity,i.e., still obtain a biological functionally equivalent protein.Hydrophilicity is another parameter that may be used to determine aminoacid substitution (see e.g., U.S. Pat. No. 4,554,101).

Exemplary amino acid substitutions that may be used in this context ofthe invention include but are not limited to exchanging arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine and isoleucine. Other such substitutionsthat take into account the need for retention of some or all of thebiological activity whilst altering the secondary structure of theprotein will be well known to those of skill in the art.

Another type of variant that is specifically contemplated for thepreparation of polypeptides according to the invention is the use ofpeptide mimetics. Mimetics are peptide-containing molecules that mimicelements of protein secondary structure. See, for example, Johnson etal., “Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto etal., Eds., Chapman and Hall, New York (1993). The underlying rationalebehind the use of peptide mimetics is that the peptide backbone ofproteins exists chiefly to orient amino acid side chains in such a wayas to facilitate molecular interactions, such as those of antibody andantigen. A peptide mimetic is expected to permit molecular interactionssimilar to the natural molecule. These principles may be used, inconjunction with the principles outlined above, to engineer secondgeneration molecules having many of the natural properties of Nav1.3protein, but with altered and even improved characteristics.

Other mutants that are contemplated are those in which entire domains ofthe Nav1.3 protein are switched with those of another related protein.For example, other sodium channels exist and chimeric sodium channelsmay be produced where domains from e.g., a Nav1.8 protein are switchedwith domains from the Nav1.3. Domain switching is well-known to those ofskill in the art and is particularly useful in generating mutants havingdomains from related species.

Domain switching involves the generation of chimeric molecules usingdifferent but related polypeptides. For example, by comparing thesequence of Nav1.3 protein with that of similar sequences from anothersource and with mutants and allelic variants of these polypeptides, onecan make predictions as to the functionally significant regions of thesemolecules. Thus, it is contemplated then to switch related domains ofthese molecules in an effort to determine the criticality of theseregions to Nav1.3 protein function. These molecules may have additionalvalue in that these “chimeras” can be distinguished from naturalmolecules, while possibly providing the same or even enhanced function.

In addition to the mutations described above, the present inventionfurther contemplates the generation of a specialized kind of insertionalvariant known as a fusion protein. This molecule generally has all or asubstantial portion of the native molecule, linked at the N- orC-terminus, to all or a portion of a second polypeptide. For example,fusions typically employ leader sequences from other species to permitthe recombinant expression of a protein in a heterologous host. Anotheruseful fusion includes the addition of an immunologically active domain,such as an antibody epitope, to facilitate purification of the fusionprotein. Inclusion of a cleavage site at or near the fusion junctionwill facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains, such as active sites from enzymes, glycosylation domains,cellular targeting signals or transmembrane regions.

There are various commercially available fusion protein expressionsystems that may be used in the present invention. Particularly usefulsystems include but are not limited to the glutathione S-transferase(GST) system (Pharmacia, Piscataway, N.J.), the maltose binding proteinsystem (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.),and the 6×His system (Qiagen, Chatsworth, Calif.). These systems arecapable of producing recombinant polypeptides bearing only a smallnumber of additional amino acids, which are unlikely to affect theantigenic ability of the recombinant polypeptide. For example, both theFLAG system and the 6×His system add only short sequences, both of whichare known to be poorly antigenic and which do not adversely affectfolding of the polypeptide to its native conformation. Another Nterminal fusion that is contemplated to be useful is the fusion of a MetLys dipeptide at the N terminal region of the protein or peptides. Sucha fusion may produce beneficial increases in protein expression oractivity.

A particularly useful fusion construct may be one in which a Nav1.3splice variant of the present invention is fused to a hapten to enhanceimmunogenicity of a Nav1.3 protein fusion construct. Such fusionconstructs to increase immunogenicity are well known to those of skillin the art, for example, a fusion of Nav1.3 protein with a helperantigen such as hsp70 or peptide sequences such as from Diptheria toxinchain or a cytokine such as IL-2 will be useful in eliciting an immuneresponse. In other embodiments, fusion construct can be made which willenhance the targeting of the Nav1.3 protein-related compositions to aspecific site or cell.

Other useful fusions include Nav1.3 protein fused to specific peptide orpolypeptide domains that serve to increase cell surface expression ofmembrane proteins. Examples of such domains include one known toincrease the cell surface expression of potassium channels byfacilitating exit from the endoplasmic reticulum (see Zerangue et alNeuron 22, 537).

Other fusion constructs including a heterologous polypeptide withdesired properties also are contemplated. Other fusion systems producepolypeptide hybrids where it is desirable to excise the fusion partnerfrom the desired polypeptide. In one embodiment, the fusion partner islinked to the recombinant Nav1.3 protein polypeptide by a peptidesequence containing a specific recognition sequence for a protease.Examples of suitable sequences are those recognized by the Tobacco EtchVirus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa (NewEngland Biolabs, Beverley, Mass.).

It will be desirable to purify Nav1.3 protein or variants thereof.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing; affinity columns specific for protein fusionmoieties; affinity columns containing Nav1.3-specific antibodies. Aparticularly efficient method of purifying peptides is fast proteinliquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur. As is common withpurification of transmembrane proteins such as ion channels, it ispossible that the purified protein may need to be incorporated insynthetic liposomes to retain biological activity.

Incorporation of proteins into liposomes is well known to those of skillin the art. The most frequently used strategy for reconstitution andcrystallization of transmembrane proteins in lipid bilayers iscomicellization of the proteins and lipids, both solubilized withdetergent, which is removed after mixing the separate solutions (e.g.,Jap et al., Ultramicroscopy. 46:45-84, 1992; Kühlbrandt, Q. Rev.Biophys. 25:1-49, 1992; Rigaud et al., Struct. Biol. 118:226-235, 1995;Mosser, Micron. 32:517-540, 2001). Additional chemical agents areusually added to the solution, including e.g., detergents such as octylthioglucoside (Scheuring et al., EMBO J. 20:3029-3035, 2001), octylglucoside, octyl glucopyranoside (Montoya et al., J. Mol. Biol.250:1-10, 1995), organic solvents such as pentane, hexane, or otherchemicals such as glucose (Walz et al., J. Mol. Biol. 282:833-845, 1998)or glycerol (Ikeda-Yamasaki et al., FEBS Lett. 425:505-508, 1998). Theconcentration of the detergent molecules in these solutions is thengradually reduced, either by dialysis or by the addition of Bio-Beads(Rigaud et al., J. Struct. Biol. 118:226-235, 1997). As theconcentration of the detergent decreases from these lipid-detergent andlipid-protein-detergent micellar solutions, lipid bilayers areprogressively formed in which the transmembrane proteins areincorporated. Usually, the morphology of the resulting 2D crystalsdepends on several poorly defined factors, and depending on thecircumstances, various structures can be obtained such as planar sheets,proteo-liposomes, multilayered stacked sheets, thin three-dimensionalcrystals, and tubes (Lacapere et al., Biophys. J. 75:1319-1329, 1998;Mosser, Micron. 32:517-540, 2001). Specifically contemplatedcompositions of the present invention include the Nav1.3 splice variantsof the present invention incorporated into a liposomal preparation usingtechniques such as those outlined above. The splice variant proteincompositions for incorporation into the liposomes may be prepared andpurified using any standard protein preparation techniques.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE. It will thereforebe appreciated that wider differing electrophoresis conditions, theapparent molecular weights of purified or partially purified expressionproducts may vary.

In addition to the full length Nav1.3 protein of SEQ ID NO:2 describedherein, smaller Nav1.3 protein-related peptides derived from thesequence of SEQ ID NO:2 and containing at least the aspartic acidresidue at the relative position number 208 of SEQ ID NO:2 (i.e., in SEQID NO:2 the aspartic acid residue in question is at position 208, inother proteins that residue may be at another position along the aminoacid sequence but it is in a position that corresponds to or is derivedfrom position 208 of SEQ DI NO:2) may be useful in various embodimentsof the present invention. Such peptides or indeed even the full lengthprotein, of the invention can also be synthesized in solution or on asolid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. See, for example, Stewart and Young,Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co., (1984); Tamet al., J. Am. Chem. Soc., 105:6442, (1983); Merrifield, Science, 232:341-347, (1986); and Barany and Merrifield, The Peptides, Gross andMeienhofer, eds, Academic Press, New York, 1-284, (1979), eachincorporated herein by reference. The Nav1.3 active protein or portionsof the protein, which correspond to the selected regions describedherein, can be readily synthesized and then screened in screening assaysdesigned to identify reactive peptides.

Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of the invention is insertedinto an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression as described herein below.

U.S. Pat. No. 4,554,101 (incorporated herein by reference) also teachesthe identification and preparation of epitopes from primary amino acidsequences on the basis of hydrophilicity. Thus, one of skill in the artwould be able to identify epitopes from within any amino acid sequenceencoded by any of the DNA sequences disclosed herein.

The protein of SEQ ID NO:2 or proteins and peptides derived thereform,may be useful as antigens for the immunization of animals relating tothe production of antibodies. It is envisioned that such protein, orportions thereof, may be coupled, bonded, bound, conjugated orchemically-linked to one or more agents via linkers, polylinkers orderivatized amino acids. This may be performed such that a bispecific ormultivalent composition or vaccine is produced. It is further envisionedthat the methods used in the preparation of these compositions will befamiliar to those of skill in the art and should be suitable foradministration to animals, i.e., pharmaceutically acceptable. Preferredagents are the carriers are keyhole limpet hemocyannin (KLH) or bovineserum albumin (BSA).

N_(AV)1.3 Splice Variant Encoding Nucleic Acids

The present invention also provides, in another embodiment, an isolatednucleic acid encoding the Nav1.3 splice variant protein of theinvention. Preferred embodiments of the present invention are directedto nucleic acid constructs comprising a sequence of SEQ ID NO:1.Preferably, the sequence is operably linked to a heterologous promoter.The present invention is not limited in scope to the particular gene(s)identified herein, however, seeing as one of ordinary skill in the artcould, using the nucleic acids corresponding to the Nav1.3 gene, readilyidentify related homologs in various other species (e.g., rat, rabbit,monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat andother species).

In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, a “Nav1.3 gene” may contain a variety of different nucleic acidbases and yet still produce a corresponding polypeptide that isfunctionally indistinguishable, and in some cases structurally, from thehuman gene disclosed herein. In preferred embodiments, the nucleic acidsencode SEQ ID NO:2. In other embodiments the nucleic acids encode afunction sodium channel alpha subunit based on the amino acid sequenceof SEQ ID NO:2 which at least comprises an aspartic acid residue at theamino acid that corresponds to the amino acid at position 208 of SEQ IDNO:2, and has an insertion of a linker between Domain 1 and Domain 2.The term “Nav1.3 gene” may be used to refer to any nucleic acid thatencodes such a protein, peptide or polypeptide and, as such, is intendedto encompass both genomic DNA, mRNA and cDNA.

Similarly, any reference to a nucleic acid should be read asencompassing a host cell containing that nucleic acid and, in somecases, capable of expressing the product of that nucleic acid. Suchcells expressing nucleic acids of the present invention are contemplatedto be particularly useful in the context of screening for agents thatinduce, repress, inhibit, augment, interfere with, block, abrogate,stimulate or enhance the function of Nav1.3 gene or protein product.Such compounds identified in these screening assay embodiments also willbe useful as sodium channel modulators of other sodium channels (e.g.,Nav1.8, Nav1.6 and the like)

Nucleic acids according to the present invention (which include genomicDNA, cDNA, mRNA, as well as recombinant and synthetic sequences andpartially synthetic sequences) may encode an entire Nav1.3 protein ofSEQ ID NO:2, or polypeptide, or allelic variant, a domain of the proteinthat expresses an activity of the wild-type sodium channel and has anaspartic acid residue at a position that corresponds to residue 208 ofSEQ ID NO:2, or any other fragment or variant of the Nav1.3 proteinsequences set forth herein, as long as those variant comprise a linkerbetween Domain 1 and Domain 2 as described herein.

The nucleic acid may be derived from genomic DNA, i.e., cloned directlyfrom the genome of a particular organism. In preferred embodiments,however, the nucleic acid would comprise complementary DNA (cDNA). Alsocontemplated is a cDNA plus a natural intron or an intron derived fromanother gene; such engineered molecules are sometime referred to as“mini-genes.” At a minimum, these and other nucleic acids of the presentinvention may be used as molecular weight standards in, for example, gelelectrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that due to the redundancy of the genetic code,a given Nav1.3 gene from a given species may be represented bydegenerate variants that have slightly different nucleic acid sequencesbut, nonetheless, encode the same protein.

As used in this application, the term “a nucleic acid encoding a Nav1.3protein” refers to a nucleic acid molecule that has been isolated fromtotal cellular nucleic acid. In preferred embodiments, the inventionconcerns a nucleic acid sequence essentially as net forth in SEQ IDNO:1. The term “functionally equivalent codon” is used herein to referto codons that encode the same amino acid, such as the six codons forarginine or serine (Table 1, below), and also refers to codons thatencode biologically equivalent amino acids, as discussed in thefollowing pages.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

Nucleotide sequences that have at least about 95% of nucleotides thatare identical to the nucleotides of the entire sequence of SEQ ID NO:1are preferred. Sequences that are essentially the same as those setforth in SEQ ID NO:1 may also be functionally defined as sequences thatare capable of hybridizing to a nucleic acid segment containing thecomplement of SEQ ID NO:1 under standard conditions.

The DNA segments of the present invention include those encodingbiologically functional equivalent Nav1.3 proteins and peptides asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through any means describedherein or known to those of skill in the art.

The present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO:1. Nucleic acid sequences that are “complementary” arethose that are capable of base-pairing according to the standardWatson-Crick complementary rules. As used herein, the term“complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1 under highlystringent conditions. Such sequences may encode the entire Nav1.3protein of SEQ ID. NO:2 or functional or non-functional fragmentsthereof.

Alternatively, the hybridizing segments may be shorter oligonucleotides.Sequences of about 17 bases long should occur only once in the humangenome and, therefore, suffice to specify a unique target sequence.Antisense nucleic acids directed against the sequence of SEQ ID NO:1 areparticularly useful.

Suitable hybridization conditions will be well known to those of skillin the art. In certain applications, it is appreciated that lowerstringency conditions may be required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

Another way of exploiting probes and primers of the present invention isin site-directed, or site-specific mutagenesis. Site-specificmutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides,through specific mutagenesis of the underlying DNA. The techniquefurther provides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids also are routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement. Site-directedmutagenesis can also be accomplished using PCR to introduce the desiredalteration in the coding sequence. In this case, one of theamplification primers contains the alteration(s) of choice and resultsin a DNA fragment containing the desired mutation(s) that can then beincorporated into the full-length construct.

Of course site-directed mutagenesis is not the only method of generatingpotentially useful mutant protein species and as such is not meant to belimiting. The present invention also contemplates other methods ofachieving mutagenesis such as for example, treating the recombinantvectors carrying the gene of interest mutagenic agents, such ashydroxylamine, to obtain sequence variants.

It will be useful to inhibit the expression of Nav1.3 to decrease theactivity of the encoded protein and effect and ameliorative outcome onpain. One may advantageously disrupt the activity or expression of aprotein using a variety of methods known to those of skill in the art.For example, nucleic acid-based methods of disrupting or block Nav1.3expression are contemplated. Polynucleotide products which are useful inthis endeavor include antisense polynucleotides, ribozymes, RNAi, andtriple helix polynucleotides that modulate the expression of Nav1.3.

Antisense polynucleotides and ribozymes are well known to those of skillin the art. Crooke and B. Lebleu, eds. Antisense Research andApplications (1993) CRC Press; and Antisense RNA and DNA (1988) D. A.Melton, Ed. Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y.Anti-sense RNA and DNA molecules act to directly block the translationof mRNA by binding to targeted mRNA and preventing protein translation.An example of an antisense polynucleotide is an oligodeoxyribonucleotidederived from the translation initiation site, e.g., between −10 and +10regions of the relevant nucleotide sequence.

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with “complementary” sequences. By complementary, it ismeant that polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarily to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozymes) could be designed. These molecules, though having lessthan 50% homology, would bind to target sequences under appropriateconditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

As indicated above, the DNA and protein sequences for the specificsplice variant of the present invention are provided in SEQ ID NO:1 andSEQ ID NO:2, respectively. Related protein and/or nucleic acid sequencesfrom other sources may be identified using probes directed at thesequences of SEQ ID NO:1. Such additional sequences may be useful incertain aspects of the present invention. Although antisense sequencesmay be full length genomic or cDNA copies, they also may be shorterfragments or oligonucleotides e.g., polynucleotides of 100 or lessbases. Although shorter oligomers (8-20) are easier to make and moreeasily permeable in vivo, other factors also are involved in determiningthe specificity of base pairing. For example, the binding affinity andsequence specificity of an oligonucleotide to its complementary targetincreases with increasing length. It is contemplated thatoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, or more base pairs will be used.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific interaction of the ribozyme molecule to complementary targetRNA, followed by an endonucleolytic cleavage. Within the scope of theinvention are engineered hammerhead or other motif ribozyme moleculesthat specifically and efficiently catalyze endonucleolytic cleavage ofRNA sequences encoding protein complex components.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures, such as secondary structure, that may render theoligonucleotide sequence unsuitable. The suitability of candidatetargets may also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays. See, Draper PCT WO 93/23569; and U.S. Pat. No.5,093,246.

Nucleic acid molecules used in triple helix formation for the inhibitionof transcription are generally single stranded and composed ofdeoxyribonucleotides. The base composition must be designed to promotetriple helix formation via Hoogsteen base pairing rules, which generallyrequire sizeable stretches of either purines or pyrimidines to bepresent on one strand of a duplex. Nucleotide sequences may bepyrimidine-based, which will result in TAT and CGC+ triplets across thethree associated strands of the resulting triple helix. Thepyrimidine-rich molecules provide base complementarity to a purine-richregion of a single strand of the duplex in a parallel orientation tothat strand. In addition, nucleic acid molecules may be chosen that arepurine-rich, for example, containing a stretch of G residues. Thesemolecules will form a triple helix with a DNA duplex that is rich in GCpairs, in which the majority of the purine residues are located on asingle strand of the targeted duplex, resulting in GGC triplets acrossthe three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Another technique that is of note for reducing or disrupting theexpression of a gene is RNA interference (RNAi), also known as smallinterfering RNA (siRNA). The term “RNA interference” was first used byresearchers studying C. elegans and describes a technique by whichpost-transcriptional gene silencing (PTGS) is induced by the directintroduction of double stranded RNA (dsRNA: a mixture of both sense andantisense strands). Injection of dsRNA into C. elegans resulted in muchmore efficient silencing than injection of either the sense or theantisense strands alone (Fire et al., Nature 391:806-811, 1998). Just afew molecules of dsRNA per cell is sufficient to completely silence theexpression of the homologous gene. Furthermore, injection of dsRNAcaused gene silencing in the first generation offspring of the C.elegans indicating that the gene silencing is inheritable (Fire et al.,Nature 391:806-811, 1998). Current models of PTGS indicate that shortstretches of interfering dsRNAs (21-23 nucleotides; siRNA also known as“guide RNAs”) mediate PTGS. siRNAs are apparently produced by cleavageof dsRNA introduced directly or via a transgene or virus. These siRNAsmay be amplified by an RNA-dependent RNA polymerase (RdRP) and areincorporated into the RNA-induced silencing complex (RISC), guiding thecomplex to the homologous endogenous mRNA, where the complex cleaves thetranscript. Thus, siRNAs are nucleotides of a short length (typically18-25 bases, preferably 19-23 bases in length) which incorporate into anRNA-induced silencing complex in order to guide the complex tohomologous endogenous mRNA for cleavage and degradation of thetranscript.

While most of the initial studies were performed in C. elegans, RNAi isgaining increasing recognition as a technique that may be used inmammalian cell. It is contemplated that RNAi, or gene silencing, will beparticularly useful in the disruption of tissue-specific geneexpression. By placing a gene fragment encoding the desired dsRNA behindan inducible or tissue-specific promoter, it should be possible toinactivate genes at a particular location within an organism or during aparticular stage of development.

Variations on RNA interference (RNAi) technology is revolutionizing manyapproaches to experimental biology, complementing traditional genetictechnologies, mimicking the effects of mutations in both cell culturesand in living animals. (McManus & Sharp, Nat. Rev. Genet. 3, 737-747(2002)). RNAi has been used to elicit gene-specific silencing incultured mammalian cells using 21-nucleotide siRNA duplexes (Elbashir etal., Nature, 411:494-498, 2001; Fire et al., Nature 391, 199-213 (1998),Harmon, G. J., Nature 418, 244-251 (2002))). In the same cultured cellsystems, transfection of longer stretches of dsRNA yielded considerablenonspecific silencing. Thus, RNAi has been demonstrated to be a feasibletechnique for use in mammalian cells and could be used for assessinggene function in cultured cells and mammalian systems, as well as fordevelopment of gene-specific therapeutics. In particularly preferredembodiments, the siRNA molecule is between 20 and 25 oligonucleotides inlength an is derived from the sequence of SEQ ID NO:1. Particularlypreferred siRNA molecules are 21-23 bases in length.

Anti-sense RNA and DNA molecules, ribozymes, RNAi and triple helixmolecules can be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides well known in the artincluding, but not limited to, solid phase phosphoramidite chemicalsynthesis. Alternatively, RNA molecules may be generated by in vitro andin vivo transcription of DNA sequences encoding the antisense RNAmolecule. Such DNA sequences may be incorporated into a wide variety ofvectors which incorporate suitable RNA polymerase promoters such as theT7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructsthat synthesize antisense RNA constitutively or inducibly, depending onthe promoter used, can be introduced stably or transiently into cells.

Commercial providers such as Ambion Inc. (Austin, Tex.), Darmacon Inc.(Lafayette, Colo.), InvivoGen (San Diego, Calif.), and Molecula ResearchLaboratories, LLC (Herndon, Va.) generate custom siRNA molecules. Inaddition, commercial kits are available to produce custom siRNAmolecules, such as SILENCER™ siRNA Construction Kit (Ambion Inc.,Austin, Tex.) or psiRNA System (InvivoGen, San Diego, Calif.). ThesesiRNA molecules may be introduced into cells through transienttransfection or by introduction of expression vectors that continuallyexpress the siRNA in transient or stably transfected mammalian cells.Transfection may be accomplished by well known methods including methodssuch as infection, calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method or other known techniques. Thesetechniques are well known to those of skill in the art.

Recombinant Protein Production.

Given the above disclosure of SEQ ID NO:1, it is possible to produce aprotein of SEQ ID NO:2 by recombinant techniques. A variety ofexpression vector/host systems may be utilized to contain and expresssuch a protein coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransfected with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.Mammalian cells that are useful in recombinant protein productioninclude but are not limited to VERO cells, HeLa cells, Chinese hamsterovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2,3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols forthe recombinant expression of a protein of amino acid sequence of SEQ IDNO:2 in bacteria, yeast and other invertebrates are described hereinbelow.

The DNA sequence encoding the mature form of the protein is amplified byPCR and cloned into an appropriate vector for example, pGEX 3X(Pharmacia, Piscataway, N.J.). The pGEX vector is designed to produce afusion protein comprising glutathione S transferase (GST), encoded bythe vector, and a protein encoded by a DNA fragment inserted into thevectors cloning site. The primers for the PCR may be generated toinclude for example, an appropriate cleavage site.

Treatment of the recombinant fusion protein with thrombin or factor Xa(Pharmacia, Piscataway, N.J.) is expected to cleave the fusion protein,releasing the proapoptotic factor from the GST portion. The pGEX3X/Nav1.3 protein construct is transformed into E. coli XL 1 Blue cells(Stratagene, La Jolla Calif.), and individual transformants wereisolated and grown. Plasmid DNA from individual transformants ispurified and partially sequenced using an automated sequencer to confirmthe presence of the desired protein-encoding gene insert in the properorientation.

Knowledge of SEQ ID NO:1 gene sequences allows for modification of cellsto permit or increase expression of endogenous Nav1.3 splice variant ofthe present invention. The cells can be modified (heterologous promoteris inserted in such a manner that it is operably linked to, e.g., byhomologous recombination) to provide increased protein expression byreplacing, in whole or in part the naturally occurring promoter with allor part of a heterologous promoter so that the cells express such aprotein at higher levels. The heterologous promoter is inserted in sucha manner that it is operably linked to gene sequence of SEQ ID NO:1.(e.g., PCT International Publication No. WO96/12650; PCT InternationalPublication No. WO 92/20808 and PCT International Publication No. WO91/09955). It is contemplated that, in addition to the heterologouspromoter DNA, amplifiable marker DNA (e.g., ada, dhfr and themultifunctional CAD gene which encodes carbamyl phosphate synthase,aspartate transcarbamylase and dihydroorotase) and/or intron DNA may beinserted along with the heterologous promoter DNA. If linked to the genesequence, amplification of the marker DNA by standard selection methodsresults in co-amplification of the Nav1.3 splice variant with the markersequence in the cells.

While certain embodiments of the present invention contemplate producingthe Nav1.3 splice variant protein using synthetic peptide synthesizersand subsequent FPLC analysis and appropriate refolding of the cysteinedouble bonds, it is contemplated that recombinant protein productionalso may be used. For example, induction of the fusion proteincontaining the protein of interest fused to GST is achieved by growingthe transformed XL 1 Blue culture at 37° C. in LB medium (supplementedwith carbenicillin) to an optical density at wavelength 600 nm of 0.4,followed by further incubation for 4 hours in the presence of 0.5 mMIsopropyl β-D Thiogalactopyranoside (Sigma Chemical Co., St. Louis Mo.).

The fusion protein, expected to be produced as an insoluble inclusionbody in the bacteria, may be purified using standard techniques in whichcells are harvested, washed, lysed and the protein extracted andpurified e.g., using the GST Purification Module (Pharmacia Biotech).The GST may be cleaved using thrombin digestion.

The recombinant protein also may be prepared using a yeast system e.g.,the Pichia Expression System (Invitrogen, San Diego, Calif.), followingthe manufacturer's instructions. Alternatively, the cDNA encoding thegiven Nav1.3 splice variant protein may be cloned into the baculovirusexpression vector pVL1393 (PharMingen, San Diego, Calif.). This vectoris then used according to the manufacturer's directions (PharMingen) toinfect Spodoptera frugiperda cells in sF9 protein free media and toproduce recombinant protein. The protein is purified and concentratedfrom the media using a heparin Sepharose column (Pharmacia, Piscataway,N.J.) and sequential molecular sizing columns (Amicon, Beverly, Mass.),and resuspended in PBS. SDS PAGE analysis shows a single band andconfirms the size of the protein, and Edman sequencing on a Porton 2090Peptide Sequencer confirms its N terminal sequence. In still otheralternatives, the an insect system expression system may be used.

Mammalian host systems for the expression of the recombinant proteinalso are well known to those of skill in the art and are most preferred.Host cell strains may be chosen for a particular ability to process theexpressed protein or produce certain post translation modifications thatwill be useful in providing protein activity. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, andthe like have specific cellular machinery and characteristic mechanismsfor such post-translational activities and may be chosen to ensure thecorrect modification and processing of the introduced, foreign protein.

It is preferable that the transformed cells are used for long-term,high-yield protein production and as such stable expression isdesirable. Once such cells are transformed with vectors that containselectable markers along with the desired expression cassette, the cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

A number of selection systems may be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoritiosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside G418; als which confers resistance tochlorsulfuron; and hygro, that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

Vectors for Cloning, Gene Transfer and Expression

As discussed in the previous section, expression vectors are employed toexpress the protein of interest, which can then be purified and, forexample, be used to vaccinate animals to generate antisera or monoclonalantibody with which further studies may be conducted.

Expression requires that appropriate signals be provided in the vectors,and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products also are provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

Throughout this application, the term “expression construct” or“expression vector” is meant to include any type of genetic constructcontaining a nucleic acid coding for gene products in which part or allof the nucleic acid encoding sequence is capable of being transcribed.The transcript may be translated into a protein, but it need not be. Incertain embodiments, expression includes both transcription of a geneand translation of mRNA into a gene product.

The nucleic acid encoding a gene product is under transcriptionalcontrol of a promoter. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the cell, or introduced syntheticmachinery, required to initiate the specific transcription of a gene.The phrase “under transcriptional control” means that the promoter is inthe correct location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter. Exemplary promoters include the human cytomegalovirus(CMV) immediate early gene promoter, the SV40 early promoter, the Roussarcoma virus long terminal repeat, β-actin, rat insulin promoter, thephosphoglycerol kinase promoter and glyceraldehyde-3-phosphatedehydrogenase promoter, all of which are promoters well known andreadily available to those of skill in the art, can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter with wellknown properties, the level and pattern of expression of the protein ofinterest following transfection or transformation can be optimized.Inducible promoters e.g., inducible ecdysone system (Invitrogen,Carlsbad, Calif.), or the Tet-Off™ or Tet-On™ system which are designedto allow regulated expression of a gene of interest in mammalian cellsalso may be used.

Modified versions of the CMV promoter that are less potent have alsobeen used when reduced levels of expression of the transgene aredesired. When expression of a transgene in hematopoietic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviruspromoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflowermosaic virus, HSV-TK, and avian sarcoma virus.

Tissue specific promoters may be used to effect transcription inspecific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues.

It is envisioned that any of the above promoters alone or in combinationwith another may be useful according to the present invention dependingon the action desired. In addition, this list of promoters should not beconstrued to be exhaustive or limiting, and those of skill in the artwill know of other promoters that may be used in conjunction with thepromoters and methods disclosed herein.

Another regulatory element contemplated for use in the present inventionis an enhancer. These are genetic elements that increase transcriptionfrom a promoter located at a distant position on the same molecule ofDNA. Enhancers are organized much like promoters. That is, they arecomposed of many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization. Enhancers useful in the present invention are well knownto those of skill in the art and will depend on the particularexpression system being employed (Scharf D et al Results Probl CellDiffer 20: 125-62, 1994; Bittner et al Methods in Enzymol 153: 516-544,1987).

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human or bovine growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

In certain embodiments of the invention, the use of internal ribosomeentry site (IRES) elements is contemplated to create multigene, orpolycistronic, messages. In specific embodiments herein it iscontemplated that host cells are created which comprise both a Nav1.3alpha subunit and one or more beta subunits. IRES elements can be linkedto heterologous open reading frames for such endeavors. IRES elementsare able to bypass the ribosome scanning model of 5′ methylated Capdependent translation and begin translation at internal sites (Pelletierand Sonenberg, Nature, 334:320-325, 1988). IRES elements from twomembers of the picornavirus family (poliovirus and encephalomyocarditis)have been described (Pelletier and Sonenberg, 1988 supra), as well anIRES from a mammalian message (Macejak and Sarnow, Nature, 353:90-94,1991). Multiple open reading frames can be transcribed together, eachseparated by an IRES, creating polycistronic messages. By virtue of theIRES element, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

There are a number of ways in which expression constructs may beintroduced into cells. In certain embodiments of the invention, theexpression construct comprises a virus or engineered construct derivedfrom a viral genome. In other embodiments, non-viral delivery such aslipid- or chemical-mediated transfection is contemplated. The ability ofcertain viruses to enter cells via receptor-mediated endocytosis, tointegrate into host cell genome and express viral genes stably andefficiently have made them attractive candidates for the transfer offoreign genes into mammalian cells (Ridgeway, In: Rodriguez R L,Denhardt D T, ed. Vectors: A survey of molecular cloning vectors andtheir uses. Stoneham Butterworth, 467 492, 1988; Nicolas and Rubenstein,In: Vectors: A survey of molecular cloning vectors and their uses,Rodriguez & Denhardt (eds.), Stoneham: Butterworth, 493 513, 1988;Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York,Plenum Press, 117 148, 1986; Temin, In: gene Transfer, Kucherlapati(ed.), New York: Plenum Press, 149 188, 1986). The first viruses used asgene vectors were DNA viruses including the papovaviruses (simian virus40, bovine papilloma virus, and polyoma) (Ridgeway, 1988 supra; Baichwaland Sugden, 1986 supra) and adenoviruses (Ridgeway, 1988 supra; Baichwaland Sugden, 1986 supra). These have a relatively low capacity forforeign DNA sequences and have a restricted host spectrum. Furthermore,their oncogenic potential and cytopathic effects in permissive cellsraise safety concerns. They can accommodate only up to 8 kb of foreigngenetic material but can be readily introduced in a variety of celllines and laboratory animals (Nicolas and Rubenstein, 1988 supra; Temin,1986 supra).

It is now widely recognized that DNA may be introduced into a cell usinga variety of viral vectors. In such embodiments, expression constructscomprising viral vectors containing the genes of interest may beadenoviral (see for example, U.S. Pat. No. 5,824,544; U.S. Pat. No.5,707,618; U.S. Pat. No. 5,693,509; U.S. Pat. No. 5,670,488; U.S. Pat.No. 5,585,362; each incorporated herein by reference), retroviral (seefor example, U.S. Pat. No. 5,888,502; U.S. Pat. No. 5,830,725; U.S. Pat.No. 5,770,414; U.S. Pat. No. 5,686,278; U.S. Pat. No. 4,861,719 eachincorporated herein by reference), adeno-associated viral (see forexample, U.S. Pat. No. 5,474,935; U.S. Pat. No. 5,139,941; U.S. Pat. No.5,622,856; U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,773,289; U.S. Pat.No. 5,789,390; U.S. Pat. No. 5,834,441; U.S. Pat. No. 5,863,541; U.S.Pat. No. 5,851,521; U.S. Pat. No. 5,252,479 each incorporated herein byreference), an adenoviral-adenoassociated viral hybrid (see for example,U.S. Pat. No. 5,856,152 incorporated herein by reference) or a vacciniaviral or a herpesviral (see for example, U.S. Pat. No. 5,879,934; U.S.Pat. No. 5,849,571; U.S. Pat. No. 5,830,727; U.S. Pat. No. 5,661,033;U.S. Pat. No. 5,328,688 each incorporated herein by reference) vector.In preferred embodiments, retroviral vectors are used to introduce theexpression construct into HEK293 cells.

Screening for Modulators of Sodium Channel Protein

The present invention also contemplates the use of human Nav1.3 splicevariant of the present invention and active fragments thereof in thescreening of compounds that modulate (increase or decrease activity) ofsodium channels. Such modulators and particularly sodium channelblockers will be useful as therapeutic agents. Assays for theidentification of these agents may make use of splice variants of theinvention in a variety of different formats and may depend on the kindof “activity” for which the screen is being conducted.

a. Assay Formats.

The present invention provides methods of screening for modulators ofhuman Nav1.3 sodium channel activity in vitro and in vivo in thepresence and absence of the candidate substance and comparing suchresults. It is contemplated that this screening technique will proveuseful in the general identification of compounds of therapeutic valueagainst e.g., pain, inflammation, and other diseases or disordersassociated with sodium channel activity. In preferred embodiments, itwill be desirable to identify inhibitors of sodium channel activity.However, in other embodiments, stimulators of such activity also may bedesirable.

In the screening embodiments, the present invention is directed to amethod for determining the ability of a candidate substance to alter thesodium channel activity of cells that either naturally express Nav1.3splice variant protein or have been engineered to express such a proteinas described herein. Alternatively, the present application teaches theuse of models for determining the in vivo effects of such compounds. Thecells or animals also may then be contacted with additional sodiumchannel blockers in combination with a putative modulator of sodiumchannel function in order to determine whether the effect of such sodiumchannel blockers is increased or decreased as a result of the presenceof the candidate substance.

An alteration in sodium channel activity, expression or processing inthe presence of the candidate substance will indicate that the candidatesubstance is a modulator of the activity.

While the above method generally describes a sodium channel splicevariant protein activity, it should be understood that candidatesubstance may be an agent that alters the expression of sodium channelprotein, thereby increasing or decreasing the amount of Nav1.3 proteinpresent as opposed to the per unit activity of the protein.

To identify a candidate substance as being capable of inhibiting proteinactivity, one would measure or determine the protein activity in theabsence of the added candidate substance. One would then add thecandidate inhibitory substance to the cell and determine the activity ofprotein in the presence of the candidate inhibitory substance. Acandidate substance which is inhibitory would decrease the sodiumchannel activity. Exemplary such assays are described below.

b. Candidate Substances.

As used herein the term “candidate substance” refers to any moleculethat is capable of modulating sodium channel Nav1.3 splice variantprotein activity or expression. The candidate substance may be a proteinor fragment thereof, a small molecule inhibitor, or even a nucleic acidmolecule. It may prove to be the case that the most usefulpharmacological compounds for identification through application of thescreening assay will be compounds that are structurally related to otherknown modulators of sodium channels. The active compounds may includefragments or parts of naturally-occurring compounds or may be only foundas active combinations of known compounds which are otherwise inactive.However, prior to testing of such compounds in humans or animal models,it will be necessary to test a variety of candidates to determine whichhave potential.

Accordingly, the active compounds may include fragments or parts ofnaturally-occurring compounds or may be found as active combinations ofknown compounds which are otherwise inactive. Accordingly, the presentinvention provides screening assays to identify agents which inhibit orotherwise treat a disorder or associated with sodium channel activity.It is proposed that compounds isolated from natural sources, such asanimals, bacteria, fungi, plant sources, including leaves and bark, andmarine samples may be assayed as candidates for the presence ofpotentially useful pharmaceutical agents.

It will be understood that the pharmaceutical agents to be screenedcould also be derived or synthesized from chemical compositions orman-made compounds. Thus, it is understood that the candidate substanceidentified by the present invention may be polypeptide, polynucleotide,small molecule inhibitors or any other inorganic or organic chemicalcompounds that may be designed through rational drug design startingfrom known agents that are used in the intervention of pain,inflammation or other diseases/disorders associated with sodium channelactivity.

The candidate screening assays are simple to set up and perform. Thus,in assaying for a candidate substance, after obtaining a cell expressingfunctional Nav1.3 splice variant protein of the invention, one willadmix a candidate substance with the cell, under conditions which wouldallow measurable sodium channel activity to occur. Exemplary sodiumchannel assays are provided below. In this fashion, one can measure theability of the candidate substance to stimulate the activity of the cellin the absence of the candidate substance. Likewise, in assays forinhibitors after obtaining a cell expressing functional Nav1.3 splicevariant protein, the candidate substance is admixed with the cell. Inthis fashion the ability of the candidate inhibitory substance toreduce, abolish, or otherwise diminish a biological effect mediated byNav1.3 splice variant protein from said cell may be detected.

“Effective amounts” in certain circumstances are those amounts effectiveto reproducibly alter sodium channel associated activity of the cell oranimal in comparison to the normal levels of such an event. Compoundsthat achieve significant appropriate changes in such activity will beused.

Significant changes in a given sodium channel activity or in vivofunction (discussed below) of at least about 30%-40%, and mostpreferably, by changes of at least about 50%, with higher values ofcourse being possible. The active compounds of the present inventionalso may be used for the generation of antibodies which may then be usedin analytical and preparatory techniques for detecting and quantifyingfurther such inhibitors.

Proteins are often used in high throughput screening (HTS) assays knownin the art, including melanophore assays to investigate receptor ligandinteractions, yeast based assay systems and mammalian cell expressionsystems. For a review see Jayawickreme and Kost, Curr. Opin. Biotechnol.8: 629 634 (1997). Automated and miniaturized HTS assays are alsocontemplated as described for example in Houston and Banks Curr. Opin.Biotechnol. 8: 734 740 (1997)

There are a number of different libraries used for the identification ofsmall molecule modulators including chemical libraries, natural productlibraries and combinatorial libraries comprised or random or designedpeptides, oligonucleotides or organic molecules. Chemical librariesconsist of structural analogs of known compounds or compounds that areidentified as hits or leads via natural product screening or fromscreening against a potential therapeutic target. Natural productlibraries are collections of products from microorganisms, animals,plants, insects or marine organisms which are used to create mixtures ofscreening by, e.g., fermentation and extractions of broths from soil,plant or marine organisms. Natural product libraries includepolypeptides, non-ribosomal peptides and non-naturally occurringvariants thereof. For a review see Science 282:63 68 (1998).Combinatorial libraries are composed of large numbers of peptidesoligonucleotides or organic compounds as a mixture. They are relativelysimple to prepare by traditional automated synthesis methods, PCRcloning or other synthetic methods. Of particular interest will belibraries that include peptide, protein, peptidomimetic, multiparallelsynthetic collection, recombinatorial and polypeptide libraries. Areview of combinatorial libraries and libraries created therefrom, seeMyers Curr. Opin. Biotechnol. 8: 701 707 (1997). A candidate modulatoridentified by the use of various libraries described may then beoptimized to modulate activity of Nav1.3 splice variant protein through,for example, rational drug design.

It will, of course, be understood that all the screening methods of thepresent invention are useful in themselves notwithstanding the fact thateffective candidates may not be found. The invention provides methodsfor screening for such candidates, not solely methods of finding them.

c. In Vitro Assays.

Those of skill in the art are aware of numerous variations of in vitromethods for measuring sodium channel activity. Cells that express thegiven sodium channel being tested e.g., a neuronal cell line (from anyeukaroyotic, preferably mammalian source) that has been transformed ortransfected with a nucleic acid that encodes a protein of SEQ ID NO:2 oralternatively, a primary mammalian cell culture e.g., neurons thatnaturally express a protein of SEQ ID NO:2 are obtained. Primary cellsfrom e.g., a rat source can be prepared as taught in Gallo et al., 1990(J. Neurochem: 54, 1619-25 or Example 155 of U.S. Pat. No. 6,756,400).The cells are plated in an appropriate support e.g., in 96-wellpoly-D-lysine-coated black wall-clear bottom culture plates at asuitable concentration (e.g., 1-2×10⁵ cells/well of a 96-well plate).The cells are maintained at 37° C. in an atmosphere containing 5% CO₂.

To measure sodium channel activity, veratridine-evoked increases inintracellular Ca²⁺ ([Ca²⁺]i) in fluo-4/AM loaded cerebellar granuleneurons may be monitored, in real-time, using a Fluorescent ImagingPlate Reader (FLIPR™, Molecular Devices, Sunnyvale, Calif.). The cellsare incubated with 4 mM fluo-4/AM in HBSS buffer containing 2.5 mMprobenecid and 0.04% pluronic acid for 45 min at 37° C. The cells arethen washed three times with HBSS containing 2.5 mM probenecid (FLIPR™buffer). The plates are transferred to the FLIPR™ and the cellsincubated for 5 min in FLIPR™ buffer, in the absence (control) orpresence of the test compound, prior to addition of veratridine (40 μM).Cell fluorescence (λ_(Ex)=488 nm; λ_(Em)=510 nm) is monitored bothbefore and after the addition of veratridine. Peak fluorescenceintensity, after veratridine addition, is determined using the FLIPR™software. Curve fitting and parameter estimation (pIC₅₀) were performedusing GraphPad. Stock solutions (10 mM) of compounds were made in 100%DMSO.

As an alternative to FLIPR™, the VIPR™ (Aurora Biosciences Corporation)assay may be used. In such an alternative assay, HEK293 cells expressinghNav1.3 channels are cultured on 96- or 384-well plates (Costar tissueculture treated 96-well flat bottom plates, Corning). To preventdetachment of cells during plate washing, these plates are pre-coatedwith 0.5% Growth Factor Reduced matrigel matrix in DMEM for 1 hour atroom temperature before use for cell culture. About 40,000 cells areseeded to each well and incubated at 38° C. for 24 hours before assay.Assay is performed at room temperature. The cell plates are first washedthree times with bath solution using automatic plate washer (ELx405,Biotek), leaving a residual volume of 50 μL/well. Subsequently, cellsare incubated with mixed dye solution for 30 min in the dark at roomtemperature. The mixed dye solution is prepared with External solutionand consists of 10 μM CC2-DMPE (chlorocoumarin-2-dimyristoylphosphatidylethanolamine), 2.4 μM DISBAC₆(3)(bis-(1,3-dihexyl-thiobarbituric acid) trimethine oxonol), 0.5%β-cyclodextrin, 20 μg/ml pluronic F-127 and ESS Acid Yellow 17 (ESSAY-17). Thereafter, the cells are washed three times again with bathsolution and then incubated with bath solution containing 0.5 mM ESSAY-17 and test compounds at desired concentrations for 10 min beforeassay.

A VIPR is equipped with instrumentation capable of electricalstimulation of cells expressing NaV1.3 (see U.S. Pat. No. 6,686,193).This allows manipulation of the membrane potential and modulates theNaV1.3 conductance. Sodium channels have brief (˜1-3 ms) open times, soa train of electric field pulses is used to cycle the channel throughopen and closed conformations repeatedly. Membrane potential changescaused by the sodium influx through the channels is converted to opticalsignals using the Aurora FRET voltage sensitive dyes, described above.Cells stained with CC2-DMPE and DiSBAC₆(3) are excited at 405 nm. Theinstrument is able to continually monitor the fluorescent output at twowavelengths for FRET measurement. Fluorescence responses are obtained attwo wavelengths, 460 nm for CC2-DMPE and 580 nm for DiSBAC₆(3).

The IC50 values of the tested compounds may be determined using an assaysuch as the one set forth above or any other conventional assay thatmeasures sodium channel activity. Compounds that are effective in suchin vitro assays may be tested in subsequent in vivo assays as describedbelow.

Such assays are highly amenable to automation and high throughput. Highthroughput screening of compounds is described in WO 84/03564.

Of particular interest in this format will be the screening of a varietyof different Nav1.3 splice variant protein mutants. These mutants,including deletion, truncation, insertion and substitution mutants, willhelp identify which domains are involved with the functional channelforming activity of the Nav1.3 splice variant of the invention. Oncethis region(s) or amino acids particularly important to the channelforming properties of the Nav1.3 splice variant protein that has thesequence of SEQ ID NO:2 has been determined, it will be possible toidentify which of these mutants have altered structure but retain someor all of the biological functions of the sodium channel. As notedabove, SEQ ID NO:2 comprises a 33-amino acid (NVSQASMSSRMVPGLPANGKMHSTVDCNGVVSL; SEQ ID NO:3) that starts at residue 624 ofthe NAV1.3 splice variant of the present invention. This region is partof the linker between domain 1 and domain 2 of the sodium channel alpha1 subunit. PICA phosphorylation sites of the sodium channel are locatedin this region and are close to this splice site. It is particularlycontemplated that amino acids in this linker region, and particularly,each of the residues in SEQ ID NO:3 will be mutated to assess the effectof such mutation on the sodium channel activity. For example, each ofthe amino acids in this domain may be separately switched to an alanineresidue and an “alanine scan” performed to determine which of theresidues is important in determining activity. In additionalembodiments, it is contemplated that each of the amino acids in thisdomain may be separately switched to another amino acid that is aconservative substitution of the native residue depending on thehyrophobicity, hydrophilicity or other characteristics of the amino acidat a given residue. To this effect, the SIFT (Sorting Intolerant FromTolerant) program is an exemplary program that allows the skilledartisan to predict whether an amino acid substitution affects proteinfunction and can distinguish between functionally neutral anddeleterious amino acid changes in mutagenesis studies (Ng and Henikoff,Nucleic Acid. Res. 31(13): 3812-3814, 2003)

Purified Nav1.3 splice variant protein or its binding agent can becoated directly onto plates for use in the aforementioned drug screeningtechniques. However, non-neutralizing antibodies to the polypeptide canbe used to immobilize the polypeptide to a solid phase. Also, fusionproteins containing a reactive region (preferably a terminal region) maybe used to link the Nav1.3 protein active region to a solid phase.

Other forms of in vitro assays include those in which functionalreadouts are taken. For example cells in which a Nav1.3 proteinpolypeptide is expressed can be treated with a candidate substance. Insuch assays, the substance would be formulated appropriately, given itsbiochemical nature, and contacted with the cell. Depending on the assay,culture may be required. The cell may then be examined by virtue of anumber of different physiologic assays, as discussed above.Alternatively, molecular analysis may be performed in which the cellscharacteristics are examined. This may involve assays such as those forprotein expression, enzyme function, substrate utilization, mRNAexpression (including differential display of whole cell or polyA RNA)and others. Yet another assay format that can be contemplated is the useof a binding assay with a suitably labeled ligand that binds to theexpressed protein. An example of such an assay would be the displacementby a small molecule of a radiolabeled or fluorescently labeled ligandfrom the expressed Nav1.3 protein. Such an assay can be used to identifypotential small molecule modulators of the channel especially if thesite where the labeled ligand binds is known to affect channel activityor regulation.

d. In Vivo Assays.

The present invention also encompasses the use of various animal models.In exemplary embodiments, the in vivo assays are set up to identifyagents that modulate the sodium channel and are effective as analgesicor anti-inflammatory agents. The ability of an agent or a combination ofagents to treat pain can be determined using known pharmacologicalmodels (for example see Kim, S. H. and Chung, J. M., Pain, 1992, 50,355-363), or using models that are similar to known models. For example,to test baseline pain responses, tests such as mechanical withdrawalfrequencies by application of different forces of calibrated von Freymonofilaments (mN: 0.24, 1.47, 4.33, 8.01, 23.69, 40.31) (Stoelting,Wood Dale, Ill.) to the plantar hind paw surface, or thermal withdrawallatencies after the application of radiant heat to the plantar hind pawsurface may be used (Mansikka et al., Exp Neurol 162: 343-349, 2000; Taoet al., Neuroscience 98: 201-206, 2000). Mechanical withdrawalfrequencies are assessed by applying calibrated von Frey monofilaments0.24 and 4.33 mN to the plantar hind paw surface (Fairbanks et al., ProcNatl Acad Sci USA 97: 10584-10589 2000; Mansikka et al., Exp Neurol 162:343-349, 2000).

Male Sprague-Dawley rats are pre-screened to determine their baseline50% withdrawal threshold using a set of von Frey filaments. The 50%withdrawal threshold for mechanical stimulation to the hind paw isdetermined by the up-down method described by Dixon W. J., Ann. Rev.Pharmacol. Toxicol., 1980, 20, 441-462. Briefly, 8 von Frey filamentswith approximately equal logarithmic incremental (0.22) bending forcesare chosen (von Frey numbers: 3.65, 3.87, 4.10, 4.31, 4.52, 4.74, 4.92,and 5.16; equivalent to: 0.45, 0.74, 1.26, 2.04, 3.31, 5.50, 8.32, and14.45 g). A von Frey filament is applied perpendicularly to the plantarsurface with sufficient force to bend it slightly and held for 3-5seconds. An abrupt withdrawal of the foot during stimulation orimmediately after the removal of stimulus is considered a positiveresponse.

Whenever there is a positive or negative response, the next weaker orstronger filament is applied, respectively. The test is continued untilsix stimuli after the first change in response has been obtained. Thepattern of positive and negative responses may then be converted into a50% threshold value using various formulae known to those of skill inthe art. One such formula is: 50% threshold=10^((X+kd))/10⁴, where X=thevalue of the final von Frey filament used (in log units), k=the tabularvalue for the pattern of positive/negative responses [obtained fromDixon, Annu Rev Pharmacol Toxicol 20:441-462], and d=the mean differencebetween stimuli in log units (0.22). In the cases where continuouspositive or negative responses are observed all the way out to the endof the stimulus spectrum, values of 0.3 g or 15.0 g are assigned,respectively. For ED₅₀ calculations, a linear regression is determinedfor responses one either side of the 50% reversal and then anapproximation is determined based upon the value which intersects the50% point.

Other in vivo methods of testing pain include hotplate analgesia meterdeterminations. Such hotplate methods evaluate the reaction time of mice(or rats) dropped onto a heated surface and confronted with a heatstimulus applied to their plantar surface. When an analgesic agent isadministered to the animals, their reaction time is markedly increased.Such methods may be assessed using e.g., the SDI Hotplate AnalgesiaMeter (San Diego Instruments, San Diego, Calif., USA).

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route that can be utilized for clinical or non-clinicalpurposes, including but not limited to oral, nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood, cerebrospinal fluid (CSF) or lymph supply andintratumoral injection.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Such criteria include, but are notlimited to, survival, reduction of tumor burden or mass, inhibition orprevention of inflammatory response, increased activity level,improvement in immune effector function and improved food intake.

Therapeutic Methods and Pharmaceutical Compositions

The present invention deals with the treatment of diseases that resultfrom the increased activity (or expression) of sodium channel proteins.Compositions that inhibit the expression or overexpression of Nav1.3protein or block its sodium channel activity will be useful in treatingor preventing a disease or condition associated with sodium channelactivity.

The phrase “disease or condition associated with sodium channelactivity” includes all disease states and/or conditions that areacknowledged now, or that are found in the future, to be associated withthe activity of sodium channels. Such disease states include, but arenot limited to, pathophysiological disorders, including hypertension,cardiac arrhythmogenesis, angina, insulin-dependent diabetes,non-insulin dependent diabetes mellitus, diabetic neuropathy, seizures,tachycardia, ischemic heart disease, cardiac failure, myocardialinfarction, transplant rejection, autoimmune disease, sickle cellanemia, muscular dystrophy, gastrointestinal disease, mental disorder,sleep disorder, anxiety disorder, eating disorder, neurosis, alcoholism,inflammation, multiple sclerosis, cerebrovascular ischemia, CNSdiseases, epilepsy, stroke, Parkinson's disease, asthma, incontinence,urinary dysfunction, micturition disorder, irritable bowel syndrome,restenosis, subarachnoid hemorrhage, Alzheimers disease, drugdependence/addiction, schizophrenia, Huntington's chorea, tension-typeheadache, trigeminal neuralgia, cluster headache, migraine (acute andprophylaxis), inflammatory pain, neuropathic pain and depression.

Nucleic acid sequences, antisense molecules, PNAs, purified protein,antibodies, antagonists or inhibitors directed against Nav1.3 can all beused as pharmaceutical compositions. Delivery of these molecules fortherapeutic purposes is further described below. The most appropriatetherapy depends on the patient, the specific diagnosis, and thephysician who is treating and monitoring the patient's condition.

Where clinical applications are contemplated, it will be necessary toprepare the small molecules, analgesic compounds, viral expressionvectors, antibodies, peptides, nucleic acids and other compositionsidentified by the present invention as pharmaceutical compositions,i.e., in a form appropriate for in vivo applications. Generally, thiswill entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the compositions of the present invention, its use intherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. The pharmaceuticalcompositions may be introduced into the subject by any conventionalmethod, e.g., by intravenous, intradermal, intramusclar, intramammary,intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary(e.g., term release); by oral, sublingual, nasal, anal, vaginal, ortransdermal delivery, or by surgical implantation at a particular site,e.g., embedded under the splenic capsule, brain, or in the cornea. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time. A “subject” or “individual” as used herein, is avertebrate, preferably a mammal, more preferably a human. Mammalsinclude research, farm and sport animals, and pets.

The active compounds may be prepared for administration as solutions offree base or pharmacologically acceptable salts in water suitably mixedwith a surfactant, such as hydroxypropylcellulose. Dispersions also canbe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms cart bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

A typical composition for intramuscular or intrathecal administrationwill consist of a suspension or solution of active ingredient in an oil,for example arachis oil or sesame oil. A typical composition forintravenous or intrathecal administration will consist of a sterileisotonic aqueous solution containing, for example active ingredient anddextrose or sodium chloride, or a mixture of dextrose and sodiumchloride. Other examples are lactated Ringer's injection, lactatedRinger's plus dextrose injection, Normosol-M and dextrose, Isolyte E,acylated Ringer's injection, and the like. Optionally, a co-solvent, forexample, polyethylene glycol; a chelating agent, for example,ethylenediamine tetracetic acid; a solubilizing agent, for example, acyclodextrin; and an anti-oxidant, for example, sodium metabisulphite,may be included in the formulation. Alternatively, the solution can befreeze dried and then reconstituted with a suitable solvent just priorto administration.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration.

In the clinical setting a “therapeutically effective amount” is anamount sufficient to effect beneficial or desired clinical results. Aneffective amount can be administered in one or more doses. The term“therapeutically effective amount” refers to an amount sufficient toeffect treatment when administered to a patient in need of treatment.The team “treatment” as used herein refers to the treatment of a diseaseor medical condition in a patient, such as a mammal (particularly ahuman) which includes: preventing the disease or medical condition fromoccurring, i.e., prophylactic treatment of a patient; ameliorating thedisease or medical condition, i.e., eliminating or causing regression ofthe disease or medical condition in a patient; suppressing the diseaseor medical condition, i.e., slowing or arresting the development of thedisease or medical condition in a patient; or alleviating the symptomsof the disease or medical condition in a patient. Thus, in terms oftreatment, a “therapeutically effective amount” of the given therapeuticagent is an amount sufficient to palliate, ameliorate, stabilize,reverse, slow or delay the progression of a disease or conditionassociated with sodium channel activity or otherwise reduce thepathological consequences of such a disease or condition. The effectiveamount is generally determined by the physician on a case-by-case basisand is within the skill of one in the art. Several factors are typicallytaken into account when determining, an appropriate dosage. Thesefactors include age, sex and weight of the patient, the condition beingtreated, the severity of the condition and the form of the antibodybeing administered.

The therapeutic compositions can also comprise one or more additionalagents effective in the treatment of a disease or disorder associatedwith sodium channel activity. Other compositions which inhibit theexpression, activity or function of Nav1.3 protein (e.g., antagonists)also are contemplated for use in such treatment methods. Thus,combination therapy for the treatment of a disease or disorderassociated with sodium channel activity is specifically contemplated. Inthe context of the present invention, it is contemplated that Nav1.3inhibition therapy could be used similarly in conjunction with otheranalgesic agents or sodium channel blocker that are used in thetreatment of such disorders.

To achieve the appropriate therapeutic outcome using the methods andcompositions of the present invention, one would generally administer afirst therapeutic agent that is a Nav1.3, inhibitor or blocker asdiscussed herein and at least one other therapeutic agent (secondtherapeutic agent). These compositions would be provided in a combinedamount effective to produce the desired therapeutic outcome. Thisprocess may involve contacting the cells with the expression constructand the agent(s) or factor(s) at the same time. This may be achieved bycontacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes the expression construct and the other includes thesecond therapeutic agent.

Alternatively, the first therapeutic agent may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the second therapeutic agent and expression constructare applied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and expression construct would still beable to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one would contact the cell with bothmodalities within about 12-24 hours of each other and, more preferably,within about 6-12 hours of each other, with a delay time of only about12 hours being most preferred. In some situations, it may be desirableto extend the time period for treatment significantly, however, whereseveral days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7or 8) lapse between the respective administrations.

Local delivery of the first therapeutic agent (i.e., the inhibitor,stimulator or other agent that decreases or increases the amount oractivity of Nav1.3 in the individual) to patients may be a veryefficient method for delivering a therapeutically effective gene tocounteract a clinical disease. Similarly, the second therapeutic agentmay be directed to a particular, affected region of the subject's body.Alternatively, systemic delivery of the first and/or second therapeuticagent may be appropriate in certain circumstances.

The active compound(s) is effective over a wide dosage range and isgenerally administered in a therapeutically effective amount. It, willbe understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

According to the invention, a compound can be administered in a singledaily dose or in multiple doses per day. The treatment regimen mayrequire administration over extended periods of time, for example, forseveral days or for from one to six weeks.

“Unit dose” is defined as a discrete amount of a therapeutic compositiondispersed in a suitable carrier. Parenteral administration may becarried out with an initial bolus followed by continuous infusion tomaintain therapeutic circulating levels of drug product. Those ofordinary skill in the art will readily optimize effective dosages andadministration regimens as determined by good medical practice and theclinical condition of the individual patient. Suitable doses of sodiumchannel blockers are in the general range of from 0.01-100 mg/kg/day,preferably 0.1-50 mg/kg/day. For an average 70 kg human, this wouldamount to 0.7 mg to 7 g per day, or preferably 7 mg to 3.5 g per day. Ingeneral, an effective amount of a compound of this invention is a dosebetween about 0.5 and about 100 mg/kg. A preferred dose is from about 1to about 60 mg/kg of active compound. A typical daily dose for an adulthuman is from about 50 mg to about 5 g.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agents and the routes of administration. The optimal pharmaceuticalformulation will be determined by one of skill in the art depending onthe route of administration and the desired dosage. See for exampleRemington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publ. Co.Easton Pa. 18042) pp 1435 1712, incorporated herein by reference. Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface areas or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein as well as thepharmacokinetic data observed in animals or human clinical trials.

Appropriate dosages may be ascertained through the use of establishedassays for determining blood levels in conjunction with relevant doseresponse data. The final dosage regimen will be determined by theattending physician, considering factors which modify the action ofdrugs, e.g., the drug's specific activity, severity of the damage andthe responsiveness of the patient, the age, condition, body weight, sexand diet of the patient, the severity of any infection, time ofadministration and other clinical factors. As studies are conducted,further information will emerge regarding appropriate dosage levels andduration of treatment for specific diseases and conditions.

In a preferred embodiment, the present invention is directed attreatment of human disorder, disease or condition associated with sodiumchannel activity, or may be alleviated by administering a sodium channelblocker or inhibitor. A variety of different routes of administrationare contemplated. For example, a classic and typical therapy willinvolve direct, injection of a discrete area.

Further Uses of Compositions of the Invention

It is contemplated that in certain diseases or disorders, the Nav1.3splice variant is overexpressed. In certain embodiments therefore,methods of diagnosing a disorder in which Nav1.3 splice variant isoverexpressed or aberrantly expressed are contemplated. Such diagnosticmethods of the present invention are achieved through the detection ofthe Nav1.3 protein or a fragment thereof that is expressed in abundanceas compared to normal expression. Such a protein may be detected usingantibodies specific for the protein in any of a number of formatscommonly used by those of skill in the art for such detection.

In another aspect, the present invention contemplates that an antibodythat is immunoreactive with any sodium channel alpha subunit may beimmunoreactive with the protein molecule of the present invention.Indeed, antibodies may be generated using the protein of SEQ ID NO:2,which specifically include antibodies that detect the 32-amino acidlinker between Domain 1 and Domain 2. Such antibodies include, but arenot limited to, polyclonal, monoclonal, chimeric, single chain, Fabfragments and fragments produced by a Fab expression library,bifunctional/bispecific antibodies, humanized antibodies. CDR graftedantibodies, human antibodies and antibodies which include portions ofCDR sequences specific for sodium channel protein of the presentinvention. Means for preparing and characterizing antibodies are wellknown in the art (see, e.g., Harlow and Lane, ANTIBODIES: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988).Antibodies specifically immunoreactive to the splice variant thatcomprises a sequence of SEQ ID NO:2 are particularly preferred.

It is proposed that antibodies specific for sodium channels will beuseful in standard immunochemical procedures, such as ELISA and Westernblot methods and in immunohistochemical procedures such as tissuestaining to determine the distribution of sodium channels.

EXAMPLE(S)

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

The present example teaches the characterization and isolation of anovel Nav1.3 cDNA clone isolated from human spinal cord RNA using RT-PCRto amplify the message in three overlapping fragments (experimentaldetails below). The three fragments were then ligated together to createthe full-length sequence. The cDNA clone isolated is a novel splicevariant that differs from previously reported Nav1.3 cDNAs (see FIG. 1).

The Nav1.3 cDNA of the present invention was used to express humanNav1.3 in cultured cell lines. The Nav1.3-expressing line was used inhigh-throughput screening to identify antagonists of Nav1.3 and has beenused to measure activity against the sodium channel.

The cell line expressing NaV1.3 was created as follows. Human EmbryonicKidney (HEK) cells were chosen as the most suitable host cell line. Thecells were transduced by retrovirus. VSV-G pseudotyped retroviralvectors were generated through a three-plasmids co-transfection. After astable pool of cells were generated from retroviral transduction, thecells were FACS (Fluorescence Activated Cell Sorter) sorted on lightscatter, selecting for individual viable cells on 96 well plates.Daughter plates were made from these plates 2 weeks after sorting andwere assayed on the EVIPR by the assay described below.

HEK293 cells expressing hNav1.3 channels are cultured on 96- or 384-wellplates (Costar tissue culture treated 96-well flat bottom plates,Corning). To prevent detachment of cells during plate washing, theseplates are pre-coated with 0.5% Growth Factor Reduced matrigel matrix inDMEM for 1 hour at room temperature before use for cell culture. About40,000 cells are seeded to each well and incubated at 38° C. for 24hours before assay. Assay is performed at room temperature. The cellplates are first washed three times with bath solution using automaticplate washer (ELx405, Biotek), leaving a residual volume of 50 μL/well.Subsequently, cells are incubated with mixed dye solution for 30 min inthe dark at room temperature. The mixed dye solution is prepared withExternal solution and consists of 10 μM CC2-DMPE(chlorocoumarin-2-dimyristoyl phosphatidylethanolamine), 2.4 μMDISBAC₆(3) (bis-(1,3-dihexyl-thiobarbituric acid) trimethine oxonol),0.5% β-cyclodextrin, 20 μg/ml pluronic F-127 and ESS Acid Yellow 17 (ESSAY-17). Thereafter, the cells are washed three times again with bathsolution and then incubated with bath solution containing 0.5 mM ESSAY-17 and test compounds at desired concentrations for 10 min beforeassay.

A VIPR is equipped with instrumentation capable of electricalstimulation of cells expressing NaV1.3 (EVIPR U.S. Pat. No. 6,686,193).This allows manipulation of the membrane potential and modulates theNaV1.3 conductance. Sodium channels have brief (˜1-3 ms) open times, soa train of electric field pulses is used to cycle the channel throughopen and closed conformations repeatedly. Membrane potential changescaused by the sodium influx through the channels is converted to opticalsignals using the Aurora FRET voltage sensitive dyes, described above.Cells stained with CC2-DMPE and DiSBAC₆(3) are excited at 405 nm. Theinstrument is able to continually monitor the fluorescent output at twowavelengths for FRET measurement. Fluorescence responses are obtained attwo wavelengths, 460 nm for CC2-DMPE and 580 nm for DiSBAC₆(3).

A. Sequence Comparison of Vertex Nav1.3 Clone Against Other Human Nav1.3sequences

FIG. 1 shows a pilup in which the sequence of the present invention (SEQID NO:2) was compared with other human clones that were identified by ablast search at NCI. The amino acid at residue 208 is aspartic acid inthe sequence identified in the present invention, however in some othersequences analyzed this residue was found to be a serine residue. Aminoacid residue 208 is in the S3-S4 linker in Domain 1 of the sodiumchannel. It is a very short linker and changes here can have asignificant effect on sodium channel activation because S4 residues mustpivot in response to changes in transmembrane voltage. Variation at thissite arises by alternative splicing and insertion of one of two exonsthat differ only at residue 208; S is found in neonatal brain NaV1.3,while D is found in adult brain. In addition, it was seen that there isan amino acid insert that starts at residue 624 of SEQ ID NO:2. Thisregion is the linker between domain 1 and domain 2. Protein kinase Aphosphorylation sites are in this region and are close to this splicesite.

B. Cloning of Nav1.3

The nucleotide sequence for a human Nav1.3 cDNA (Genbank accession#AJ251507) was used to establish oligonucleotide primer sequences andRT-PCR strategies for the cloning of a full-length hNav1.3 cDNA. Firststrand cDNA synthesis (Clontech, Advantage RT-for-PCR, #639505) wascarried out using 100 ng of whole brain RNA (Clontech, #636530) withthree Nav1.3 specific antisense oligos (X1-26α, X2-22α and X3-29α, 1 μlof 20 μM for each oligonucleotide) in a total of reaction volume of 20μl at 42° C. for 45 minutes.

An hNav1.3 cDNA was subsequently subcloned in three fragments (A, B andC) overlapping at unique internal restriction sites (Nsi I and Bgl II)using the first-strand cDNA as template and nested PCR. The individualPCR reactions were carried out initially using an outer set of primers(X1-20s+X1-26α, X2-22s+X2-22α, X3-22s+X3-29α) and the reaction productssubsequently used as template with an inner set of oligonucleotideprimers (X1-32s+X1-25α, X2-20s+X2-21α, X3-21s+X3-) for further PCRamplification and isolation of the three Nav1.3 overlapping fragments.Restriction sites were inserted into RT-PCR oligonucleotides at the 5′(Xho I) and 3′ (Not I) ends. PCR reaction conditions for both the outerand inner set of oligonucleotides was carried out for 15 cycles of 1′ at95° C., 1′ at 55° C. and 6′ at 72° C. and then another 15 cycles of 1′at 95° C., 1′ at 58° C. and 6′ at 72° C. Reaction samples were run on 1%agarose gels, the expected molecular weight band sizes were isolated(frag A=1.2 kb, frag B=1.37 kb and frag C=2.45 kb), subsequentlyblunt-end ligated into the TOPO II vector (Invitrogen) and individualminiprep DNA samples of the fragments were sequenced.

The three fragments were then subcloned in sequence into the vectorpLBCX vector, which is derived from the pLNCX vector (Clontech) butreplaces the gene for neomycin resistance with one providing resistanceblasticidin. The completed expression vector pLBChNav1.3, containing anovel human Nav1.3 splice product, was utilized to generate retroviralsupernatants for infection of HEK293 cells. The infected HEK293 cellswere selected in 5 ug/ml blasticidin for two weeks and then single cellswere FACSorted for isolation of distinct clonal cell lines, which weretested for Nav channel expression using voltage-sensitive optical dyesand electrical stimulation on eVIPR. Clonal lines with the expecteddepolarizing responses were further subjected to patch clamp recordingtechniques and pharmacological characterization on eVIPR. Clonal lineswere chosen and an eVIPR assay protocol optimized as described above.

Table of Oligonucleotide Primers: X1-20s CTA CAC GTA ATT AAA TGT GC(SEQ ID NO: 9) X1-26α AAT GGA TCC ATA ACA ATT AAA TTC  AC(SEQ ID NO: 10) X1-32s NNN NNC TCG AGA GGA TGA AAA GAT   GGC ACA GGC(SEQ ID NO: 11) X1-25α GTT TTA CTT TTA ACC ATG CAT CAC (SEQ ID NO: 12)X2-22s ATT TGC CAA TGT GTT CTT GAT C (SEQ ID NO: 13) X2-22αTTG TGC TCA ACA ATA CTG TAG C (SEQ ID NO: 14) X2-20sCTG GGA CTG CTG TGA TGC AT (SEQ ID NO: 15) X2-21αGAA GAT TCC ACC AGA TCT TCC (SEQ ID NO: 16) X3-22sAGA AGA CCT GTC AAG TAA GTA C (SEQ ID NO: 17) X3-29αCA CAA AGA TAA TTC TTT GTT TCT   TTT TAC (SEQ ID NO: 18) X3-21sGAA GAA GGC AAA GGG AAG ATC (SEQ ID NO: 19) X3-NNN NNG CGG CCG CTT TTT ACT TTT   GAT TTT CTC TGA CC (SEQ ID NO: 20)

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

The references cited herein throughout, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are all specifically incorporated herein by reference.

1. An isolated recombinant nucleic acid encoding a human sodium channelNa_(V)1.3 polypeptide wherein said polypeptide is encoded by the nucleicacid sequence presented in SEQ ID NO:
 1. 2. An isolated recombinantnucleic acid encoding a human sodium channel Na_(v)1.3 recombinantprotein having the amino acid sequence of SEQ ID NO:
 2. 3. An isolatedrecombinant nucleic acid comprising the sequence presented in SEQ ID NO:1, the mature protein coding portion thereof, or a complement thereof.4. An isolated recombinant nucleic acid encoding a polypeptide of SEQ IDNO:
 2. 5. The isolated nucleic acid of any of claim 1, 2, 3 or 4,wherein said nucleic acid is genomic DNA.
 6. The isolated nucleic acidof any of claim 1, 2, 3 or 4, wherein said nucleic acid is cDNA.
 7. Anisolated nucleic acid comprising a nucleotide sequence encoding apolypeptide that is a conservative variant of the amino acid sequenceset forth in SEQ ID NO:2, wherein said variant encodes a sodium channelα-subunit with the proviso that residue 208 of SEQ ID NO:2 is anaspartic acid residue and an insert of 33 amino acids is found afterresidue 623, as defined in SEQ ID NO:2.
 8. An expression constructcomprising an isolated nucleic acid encoding a protein having an aminoacid sequence of SEQ ID NO:2 or the mature protein portion thereofwherein said mature protein region comprises an aspartic acid residue atthe residue that corresponds to amino acid residue 208 in SEQ ID NO:2,an insert of 33 amino acids is found after residue 623, as defined inSEQ ID NO:2 and a promoter operably linked to said polynucleotide. 9.The expression construct of claim 8, wherein said nucleic acid comprisesa mature protein coding sequence as set forth in SEQ ID NO:1.
 10. Theexpression construct of claim 8, wherein said expression construct is anexpression construct selected from the group consisting of anadenoassociated viral construct, an adenoviral construct, a herpes viralexpression construct, a vaccinia viral expression construct, aretroviral expression construct, a lentiviral expression construct and anaked DNA expression construct.
 11. A recombinant host cell stablytransformed or transfected with a nucleic acid of any of claims 1through 7, in a manner allowing the expression in said host cell of aprotein of SEQ ID NO:2.
 12. The recombinant host cell of claim 11,wherein said nucleic acid comprises a mature protein encoding sequenceas set forth in SEQ ID NO:1, wherein said mature protein encoded by saidsequence is a sodium channel Na_(V)1.3 polypeptide that has an asparticacid residue at the amino acid that corresponds to amino acid residue208 of SEQ ID NO:2 and an insert of 33 amino acids is found afterresidue 623, as defined in SEQ ID NO:2.
 13. A recombinant host cellstably transformed or transfected with an expression construct of claim8, in a manner allowing the expression in said host cell of a proteinproduct of the expression construct.
 14. The recombinant host cell ofclaim 11, wherein said host cell is a mammalian cell, a bacterial cell,a yeast cell, or an insect cell.
 15. The recombinant host cell of claim11, wherein said host cell further expresses a β-subunit of a sodiumchannel selected from the group consisting of β1, β2, β3 and β4.
 16. Therecombinant host cell of claim 11, wherein said host cell is a HEK293cell line.
 17. An isolated and purified protein comprising an amino acidsequence selected from the group consisting of an amino acid sequenceset forth in SEQ ID NO:2 and the mature protein portion of SEQ ID NO:2,wherein the mature protein portion comprises an aspartic acid residue atthe amino acid residue that corresponds to amino acid residue 208 of SEQID NO:2 and an insert of 33 amino acids is found after residue 623, asdefined in SEQ ID NO:2.
 18. An isolated and purified protein comprisingan amino acid sequence that is 99% identical to the complete sequenceset forth in SEQ ID NO:2.
 19. A diagnostic kit for detecting a nucleicacid that encodes a sodium channel α-subunit polypeptide, thepolypeptide being encoded by the sequence presented in SEQ ID NO: 1,comprising an isolated nucleic acid probe complementary to the completesequence of SEQ ID NO: 1, and means for containing said nucleic acid.20. A method of identifying a modulator of a human sodium channelα-subunit expression or activity identified by a method comprising thesteps of: i) contacting a cell that expresses a nucleic acid of SEQ IDNO:1 with the candidate modulator substance; ii) monitoring theexpression or ion channel activity of a protein of SEQ ID NO:2; and iii)comparing the expression or ion channel activity of a protein of SEQ IDNO:2 in the presence and absence of said candidate substance; wherein analteration in the expression or ion channel activity of a protein of SEQID NO:2 indicates that the substance is a modulator of human sodiumchannel α-subunit expression or activity.
 21. The method of claim 20wherein the modulator of human sodium channel α-subunit expression oractivity comprises a small molecule ion channel blocker or inhibitor, anoligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, anRNA oligonucleotide, an RNA oligonucleotide having at least a portion ofsaid RNA oligonucleotide capable of hybridizing with RNA to form anoligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 22. A methodof identifying a test compound that binds to a sodium channelcomprising: i) providing a cell that expresses a sodium channel having asequence of SEQ ID NO:2; ii) contacting the host cell with said testcompound and determining the binding of said test compound to the sodiumchannel; and iii) comparing the binding of the test compound to the hostcell determined in step (b) to the binding of said test compound with acell that does not express a sodium channel.
 23. An assay method foridentifying a test compound that modulates the activity of a sodiumchannel comprising: i) providing a host cell that expresses a functionalsodium channel comprising at least one polypeptide comprising the aminoacid sequence of SEQ ID NO: 2, (ii) contacting the host cell with a testcompound under conditions that would activate sodium channel activity ofsaid functional sodium channel in the absence of the test compound; and(iii) determining whether the host cell contacted with the test compoundexhibits a modulation in activity of the functional sodium channel. 24.The assay method of claim 22, wherein the host cell has been geneticallyengineered to express or overexpress the functional sodium channel. 25.The assay method of claim 22, wherein the host cell has been geneticallyengineered by the introduction into the cell of a nucleic acid moleculehaving a nucleotide sequence encoding a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:2.
 26. The assay method of claim22, wherein the host cell has been genetically engineered to upregulatethe expression of a nucleic acid encoding a polypeptide comprising theamino acid sequence of SEQ ID NO:
 2. 27. The assay method of claim 26,wherein the upregulated nucleic acid is endogenous to the host cell. 28.The assay method of claim 21, wherein the modulation of the functionalsodium channel activity is an inhibition of that activity.
 29. A methodof producing a purified human sodium channel α-subunit proteincomprising i) preparing an expression construct comprising a nucleicacid of SEQ ID NO:1 operably linked to a promoter; ii) transforming ortransfecting a host cell with said expression construct in a mannereffective to allow the expression of a protein having an amino acidsequence of SEQ ID NO:2, or the mature protein portion thereof in saidhost cell; iii) culturing said transformed or transfected cell underconditions to allow the production of said protein by said transformedor transfected host cell; and iv) isolating the human sodium channelα-subunit protein from said host cell.
 30. A method of treating adisorder comprising administering to a subject in need thereof apharmaceutical composition that comprises a compound identifiedaccording to any of 20-28 and a pharmaceutically acceptable carrier,excipient or diluent.